Reactive oxygen species-independent activation of the IL-1beta inflammasome in cells from patients with chronic granulomatous disease

Abstract

Humans with chronic granulomatous diseases (CGDs) due to mutations in p47-phox have defective NADPH activity and thus cannot generate NADPH-dependent reactive oxygen species (ROS). The role of ROS in inflammation is controversial; some in vitro studies suggest that ROS are crucial for secretion of IL-1beta via inflammasome activation, whereas mice defective for ROS and patients with CGD have a proinflammatory phenotype. In this study, we evaluated activation of the IL-1beta inflammasome in cells from CGD patients. In contrast to previous studies using the small molecule diphenylene iodonium (DPI) as a ROS inhibitor, we found no decrease in either caspase-1 activation or secretion of IL-1beta and IL-18 in primary CGD monocytes. Moreover, activation of CGD monocytes by uric acid crystals induced a 4-fold higher level of IL-1beta secretion compared with that seen in monocytes from unaffected subjects, and this increase was not due to increased synthesis of the IL-1beta precursor. In addition, Western blot analysis of CGD cells revealed that caspase-1 activation was not decreased, but rather was increased compared with control cells. Examination of the effects exerted by the inhibition of ROS activity by DPI revealed that the decrease in IL-1beta secretion by DPI was actually due to inhibition of IL-1beta gene expression. Thus, inconsistent with the proinflammatory role of ROS, the present findings support the concept that ROS likely dampen inflammasome activation. The absence of ROS in CGD monocytes may explain the presence of an inflammatory phenotype characterized by granulomas and inflammatory bowel disease occurring in CGD patients.

Fig. 1.

ROS inhibition decreases production and transcription of IL-1β and TNF-α. (A) Monocytes isolated from eight healthy controls were stimulated with LPS in the absence or presence of the ROS inhibitor DPI. In the presence of DPI, IL-1β production was completely inhibited (n = 8). (B) TNF-α production also was decreased when LPS-stimulated PBMCs of healthy controls were cultured in the presence of DPI (n = 2). (C) PBMCs of healthy controls were stimulated for 4 h with LPS in the absence or presence of DPI. mRNA was isolated from the cell lysates using TRIzol. DPI decreased mRNA expression of IL-1β (n = 6). (D) DPI also decreased mRNA expression of TNF-α (n = 2). (E) Active p10 caspase-1 was still expressed in cells cultured in the presence of DPI. Data are representative for four healthy volunteers.

Fig. 2.

Inflammasome activation and IL-1β production in CGD patients. (A) Monocytes isolated from CGD patients and healthy controls produced IL-1β on stimulation with LPS, Pam3cys, and Candida. (B) IL-1β stimulation by LPS and ATP was similar in CGD and control individuals. Data are presented as mean ± SEM of five healthy controls and of three CGD patients.

Fig. 3.

ROS inhibit inflammasome activation. (A) Active p10 caspase-1 was expressed more strongly in unstimulated monocytes from CGD patients than in those from healthy controls. (B) NALP3 inflammasome stimulus uric acid crystals stimulated IL-1β release in monocytes isolated from CGD patients, but not in those from healthy controls. Data are presented as mean ± SEM of five healthy controls and of three CGD patients.

Fig. 4.

ROS inhibition decreased IL-1β production in both healthy controls and CGD patients. In both healthy controls and CGD patients, DPI decreased IL-1β production in LPS-stimulated cells. Data are presented as mean ± SEM.