Inflammasome activation in NADPH oxidase defective mononuclear phagocytes from patients with chronic granulomatous disease

Abstract

Chronic granulomatous disease (CGD) is IntroductionChronic granulomatous disease (CGD) is a genetically heterogeneous primary immunodeficiency caused by defects in phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits. 1 This phagocyte oxidase generates superoxide by transferring electrons from NADPH to molecular oxygen and consists of the catalytic subunit gp91phox, structurally stabilized by p22phox, and of the regulatory subunits p47phox, p40phox, p67, and RAC. 2 Loss-of-function mutations in any of these components abrogate oxidase activity and compromise host immunity against certain bacteria and fungi. However, there is now increasing evidence for excessive inflammation in CGD even in the absence of infectious agents with increased frequency and severity of granulomatous inflammatory reactions, notably colitis. [3][4][5] Inflammasomes are implicated in host protection and a variety of inflammatory diseases by regulating the maturation of the caspase-1 dependent cytokines interleukin-1␤ (IL-1␤) and IL-18 in response to a broad range of danger signals. 6 However, the function of the phagocyte oxidase during caspase-1-mediated proinflammatory responses is controversial, and the implications of such responses for CGD pathology are poorly understood. 7 Here we evaluated the contribution of the phagocyte oxidase to the exuberant inflammatory responses associated with CGD, by analyzing inflammasome activation in cells from CGD patients. We show that mononuclear phagocytes from CGD patients activate caspase-1 and produce biologically active IL-1␤ in response to danger signals. Notably, IL-1 release from monocytes was elevated in CGD patients and could be controlled by IL-1 receptor antagonist (IL-1Ra) ex vivo and during treatment with anakinra. Our results implicate caspase-1-mediated inflammation in CGD pathogenesis and may help designing therapies to ameliorate inflammatory conditions in these patients. Methods Human monocytes and macrophagesAll experiments with human samples were done after informed consent was received from the patients and parents in accordance with the Declaration of Helsinki, as part of a protocol approved by the institutional review board of the University Children's Hospital Zurich. Blood from CGD patients, heterozygous carriers, and healthy donors (supplemental Tables 1-2, available on the Blood Web site; see the Supplemental Materials link at the top of the online article) was drawn on ethylenediaminetetraacetic acid (EDTA) and diluted in phosphatebuffered saline (PBS) supplemented with 2mM EDTA, pH 7.2 (PBS/EDTA). Up to 30 mL of blood were layered on 15 mL of Histopaque (density ϭ 1.077 g/mL) and centrifuged at 400g and 21°C for 30 minutes. The resulting interphase containing peripheral blood mononuclear cells (PBMCs) was isolated and washed with PBS/EDTA at 400g and 21°C for 10 minutes. Platelets were removed by repeated centrifugation at 200g for 15 minutes. Monocytes were isolated from PBMCs by depletion of nonmonocytes (negative selection) using the Monocyt...

Monosodium urate crystals trigger Nrf2- and heme oxygenase-1-dependent inflammation in THP-1 cells

“…Either NADPH oxidase or cyclooxygenase-2 (COX-2) is believed to contribute to ROS production in response to NLRP3-mediated inflammation. 15,16 The expression levels of p47phox, a subunit of NADPH oxidase and of COX-2 were analyzed by western blotting techniques. The levels of cytosolic p47phox and COX-2 proteins were increased in response to MSU in a dosedependent manner (Supplementary Figure 1).…”

Phagocyte NADPH Oxidase Restrains the Inflammasome in ANCA-Induced GN

“…31 More recently, it was shown in patients that Phox deficiency caused increased caspase-1 activity and IL-1b generation. 32,33 However, this issue remains a matter of debate, because several groups showed that ROS had either no effect or an accelerating effect on inflammasome-mediated IL-1b. [34][35][36] Furthermore, the ROS source and the specific ROS molecule that controls inflammasome activity are not yet characterized.…”

NADPH oxidase controls neutrophilic response to sterile inflammation in mice by regulating the IL-1α/G-CSF axis

“…IL-1b when stimulated in vitro with particulate and soluble activators of caspase-1 and NLRP3 inflammasome, 44,47,48 and 1 study reported increased IL-1a release by LPS-stimulated human CGD monocytes. 47 Here, we showed that NADPH oxidase deficiency augmented IL-1a and IL-1b release from murine resident macrophages, BMDMs, and BMDCs challenged with classic DAMPs that activate the NLRP3 inflammasome. However, we found that IL-1b played little role in vivo in driving excessive acute-phase inflammation in response to DAMPassociated tissue injury in CGD.…”