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

Abstract

Chronic granulomatous disease (CGD) is an inherited disorder characterized by recurrent infections and deregulated inflammatory responses. CGD is caused by mutations in subunits of the NADPH oxidase, an enzyme that generates reactive oxygen species in phagocytes. To elucidate the contribution of the proinflammatory protease caspase-1 to aberrant inflammatory reactions in CGD, we analyzed cells isolated from patients with defects in the phagocyte oxidase subunits p22phox, p47phox or gp91phox. We report that mononuclear phagocytes from CGD patients activated caspase-1 and produced biologically active interleukin-1beta (IL-1beta) in response to danger signals. Notably, caspase-1 activation and IL-1beta secretion from CGD monocytes was elevated in asymptomatic patients and strongly increased in patients with noninfectious inflammatory conditions. Treatment with IL-1 receptor antagonist reduced IL-1 production in monocytes ex vivo and during medical therapy. Our results identify phagocyte oxidase defective monocytes as a source of elevated IL-1 and provide a potential therapeutic option to ameliorate inflammatory conditions associated with CGD.

Figure 1

Macrophages from CGD patients activate caspase-1 and secrete mature IL-1β. (A-B) Caspase-1 activation in monocyte-derived macrophages determined by a fluorescent inhibitor of active caspase-1 (FLICA). (A) Lipopolysaccharide (LPS)–primed (−) macrophages from a chronic granulomatous disease (CGD) patient (p22) and a healthy control (C1) stimulated for 1 hour with adenosine triphosphate (ATP) or for 6 hours with silica crystals (SiO₂) or monosodium urea (MSU) crystals. Numbers above bracketed lines indicate percentage of cells with active caspase-1. (B) Active caspase-1 in macrophages from 3 CGD patients with the indicated mutations and 3 healthy controls (C1-C3) quantified by caspase-1 FLICA. (C-E) Macrophages from the indicated CGD patients and healthy donors stimulated with LPS plus ATP, nigericin (NI), SiO₂, or MSU. The production of mature interleukin-1β (IL-1β) at the indicated time points was determined by enzyme-linked immunosorbent assay. Data are representative of 6 experiments with cells from at least 5 different CGD patients (error bars indicate SEM of triplicate wells).

Figure 2

Elevated IL-1 secretion from CGD monocytes can be counteracted with IL-1Ra. (A-B) Caspase-1 activation in monocytes determined by caspase-1 FLICA. (A) Unstimulated (−) and LPS-treated (6 hours) monocytes from an asymptomatic CGD patient (p47) and a healthy control (C1). (B) Active caspase-1 in monocytes from 2 asmyptomatic CGD patients with the indicated mutation and 2 healthy controls (C1, C2) quantified by caspase-1 FLICA. (C-D) IL-1β (C) and IL-1α (D) release from monocytes of 2 asymptomatic CGD patients and a healthy control (C1) treated with LPS for the indicated time points determined by enzyme-linked immunosorbent assay. (E) Caspase-1 activation in unstimulated and LPS-treated (6 hours) monocytes from a symptomatic CGD patient with colitis determined by caspase-1 FLICA. (F-G) IL-1β (F) and IL-1α (G) release from monocytes of the indicated symptomatic CGD patient before (pre-anakinra) and after (post-anakinra) treatment with anakinra compared with a healthy control (C1). Data are representative of 4 (A-D) or 3 (E) experiments with cells from at least 2 different CGD patients (error bars indicate SEM of triplicate wells).