Abstract
Chronic granulomatous disease (CGD) is caused by mutations in genes that encode the NADPH-oxidase and result in a failure of phagocytic cells to produce reactive oxygen species (ROS) via this enzyme system. Patients with CGD are highly susceptible to infections and often suffer from inflammatory disorders; the latter occurs in the absence of infection and correlates with the spontaneous production of inflammatory cytokines. This clinical feature suggests that NADPH-oxidase-derived ROS are not required for, or may even suppress, inflammatory processes. Experimental evidence, however, implies that ROS are in fact required for inflammatory cytokine production. By using a myeloid cell line devoid of a functional NADPH-oxidase and primary CGD cells, we analyzed intracellular oxidants, signs of oxidative stress, and inflammatory cytokine production. Herein, we demonstrate that phagocytes lacking a functional NADPH-oxidase, namely primary CGD phagocytes and a gp91phox-deficient cell line, display elevated levels of ROS derived from mitochondria. Accordingly, these cells, despite lacking the major source of cellular ROS, display clear signs of oxidative stress, including an induced expression of antioxidants and altered oxidation of cell surface thiols. These observed changes in redox state were not due to abnormalities in mitochondrial mass or membrane integrity. Finally, we demonstrate that increased mitochondrial ROS enhanced phosphorylation of ERK1/2, and induced production of IL8, findings that correlate with previous observations of increased MAPK activation and inflammatory cytokine production in CGD cells. Our data show that elevated baseline levels of mitochondria-derived oxidants lead to the counter-intuitive observation that CGD phagocytes are under oxidative stress and have enhanced MAPK signaling, which may contribute to the elevated basal production of inflammatory cytokines and the sterile inflammatory manifestations in CGD.
Highlights
In response to exogenous stimuli and endogenous danger signals, phagocytes assemble the NADPH-oxidase to generate vast amounts of highly reactive oxidants, called reactive oxygen species (ROS) [1, 2]
phorbol myristate acetate (PMA) caused an increase in Dichlorofluorescin diacetate (DCFDA) fluorescence in PLB phagocytes (DCFDA reacting with induced NADPHoxidase-derived ROS (phoxROS)), but not X-PLB cells (Figure 1C)
To provide mechanistic evidence to link our observed correlation between the elevated production of both pro-inflammatory cytokines and Mitochondrial-derived ROS (mtROS) in phoxROS-deficient cell lines and primary phagocytes, we investigated the ability of Antimycin A, a specific inducer of mtROS (Figure 3A; Figure S2 in Supplementary Material), to directly induce intracellular signaling via phosphorylation of ERK1/2, and subsequent pro-inflammatory cytokine production (IL8)
Summary
In response to exogenous stimuli and endogenous danger signals, phagocytes assemble the NADPH-oxidase to generate vast amounts of highly reactive oxidants, called reactive oxygen species (ROS) [1, 2]. This stimulus-induced, rapid production of NADPH-oxidase-derived ROS ( called phoxROS) is commonly referred to as the phagocytic oxidative burst and is imperative for the oxidative killing of microbes. CGD patients frequently suffer from severe and debilitating inflammatory conditions [5] This aspect of the disease is not well understood but correlates with enhanced basal and stimulus-induced inflammatory signaling and proinflammatory cytokine production by CGD immune cells [6,7,8,9,10]
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