IL-1 receptor blockade restores autophagy and reduces inflammation in chronic granulomatous disease in mice and in humans

Abstract

Patients with chronic granulomatous disease (CGD) have a mutated NADPH complex resulting in defective production of reactive oxygen species; these patients can develop severe colitis and are highly susceptible to invasive fungal infection. In NADPH oxidase-deficient mice, autophagy is defective but inflammasome activation is present despite lack of reactive oxygen species production. However, whether these processes are mutually regulated in CGD and whether defective autophagy is clinically relevant in patients with CGD is unknown. Here, we demonstrate that macrophages from CGD mice and blood monocytes from CGD patients display minimal recruitment of microtubule-associated protein 1 light chain 3 (LC3) to phagosomes. This defect in autophagy results in increased IL-1β release. Blocking IL-1 with the receptor antagonist (anakinra) decreases neutrophil recruitment and T helper 17 responses and protects CGD mice from colitis and also from invasive aspergillosis. In addition to decreased inflammasome activation, anakinra restored autophagy in CGD mice in vivo, with increased Aspergillus-induced LC3 recruitment and increased expression of autophagy genes. Anakinra also increased Aspergillus-induced LC3 recruitment from 23% to 51% (P < 0.01) in vitro in monocytes from CGD patients. The clinical relevance of these findings was assessed by treating CGD patients who had severe colitis with IL-1 receptor blockade using anakinra. Anakinra treatment resulted in a rapid and sustained improvement in colitis. Thus, inflammation in CGD is due to IL-1-dependent mechanisms, such as decreased autophagy and increased inflammasome activation, which are linked pathological conditions in CGD that can be restored by IL-1 receptor blockade.

Keywords: Crohn disease; LPS; S. aureus; autoinflammatory disease; interleukin-1.

Fig. 1.

Effects of ROS deficiency on autophagy and IL-1β release from murine macrophages and human monocytes. (A) Mean ± SEM percentage of colocalization of LC3 with E. coli (strain LF82) in peritoneal macrophages from WT and p40^phox−/−mice after 30 min of exposure. (B) Mean ± SEM percentage of LC3 colocalized with S. aureus in monocytes from HCs and patients with CGD following a 1-h exposure to FITC-labeled S. aureus. Data are representative of one of two separate observations in two patients with CGD. (C) Mean ± SEM. IL-1β was secreted from BMMs of WT and p40^phox−/−mice stimulated with 100 ng/mL LPS in the absence (open bars) or presence (filled bars) of 10 nM 3MA. (D) IL-1β released from PBMCs of four patients with CGD and four HCs under the same conditions described in C. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

Blocking IL-1R in p47^phox−/−mice with colitis and invasive aspergillosis. C57BL/6 or p47^phox−/−mice received 2.5 mg of TNBS dissolved in 50% (vol/vol) ethanol given intrarectally and were treated i.p. with daily anakinra (10 mg/kg). Control mice received 50% ethanol. (A) Mean ± SEM weight change in two experiments. dpi, days postinfection. (B) Mean ± SEM histological score of the colon from one of two experiments. (C and D) C57BL/6 or p47^phox−/−mice were infected intranasally with live A. fumigatus conidia and treated with daily anakinra (1 or 10 mg/kg). (C) Mean ± SEM percentage of survival. (D) Mean ± SEM log₁₀ cfu in the lungs of infected mice (day 7). (E) Mean ± SEM cytokine levels in lung homogenates. (F) Mean ± SEM fold difference in MPO mRNA levels in the lungs at day 7. Data are representative of three experiments with four to eight mice per group. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

Anakinra inhibits inflammasome activation and restores autophagy in p47^phox−/−mice with invasive aspergillosis. (A) Western blotting of pro- and processed caspase-1 in lung homogenates on day 20 following infection with live A. fumigatus in untreated (None) and anakinra-treated mice compared with uninfected mice (Naive). β-tub, β-tubulin. (B) Density of bands from A. Data are representative of three experiments. (C) Mean percentage of LC3⁺ cells in EGFP-LC3–transfected RAW 264.7 cells exposed to A. fumigatus SC in the absence (None) or presence of anakinra (1 or 10 μg/mL) or rapamycin (50 μM) for 4 h (n = 3). **P < 0.01 (anakinra- or rapamycin-treated vs. untreated cells). (D) Western blot of LC3b and p62 in homogenates of lungs of naive, Aspergillus-infected untreated, or Aspergillus-infected anakinra-treated (10 mg/kg) mice on day 7 postinfection. (E) Density of bands from D. (F) LC3 staining in alveolar macrophages from C57BL/6 and p47^phox−/−mice exposed to 20 μg/mL poly(I:C) (Sigma–Aldrich), A. fumigatus SC, and/or 10 μg/mL anakinra for 2 h. (Magnification: 100×.) DAPI was used to detect nuclei. One experiment representative of two experiments is shown.

Fig. 4.

Anakinra restores autophagy in human CGD cells and reduces disease severity in patients with CGD colitis. (A) Percentage of colocalization of LC3 with resting conidia (RC) or SC in PBMCs isolated from two patients with CGD and two HCs. (B) Percentage of colocalization of LC3 with RC or SC in the same PBMCs isolated from two patients with CGD in the absence or presence of anakinra (10 μg/mL). Data are representative of two separate experiments performed in PBMCs from two patients with CGD. *P < 0.05; **P < 0.01. (C) Two patients with CGD (P1 and P2) with active colitis were treated with anakinra at a dosage of 100 mg daily for 3 mo. C-reactive protein (CRP) (milligrams per liter) and the number of stools per day are shown. (D) Patient 2 (P2). The number of perirectal abscesses during a 3-mo period of treatment with anakinra is shown.