Lysosomal storage and impaired autophagy lead to inflammasome activation in Gaucher macrophages

Abstract

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylcer-amide macrophages, the accumulation of glucosylceramide in lysosomes and the secretion of inflammatory cytokines. However, the connection between this lysosomal storage and inflammation is not clear. Studying macrophages derived from peripheral monocytes from patients with type 1 Gaucher disease with genotype N370S/N370S, we confirmed an increased secretion of interleukins IL-1β and IL-6. In addition, we found that activation of the inflammasome, a multiprotein complex that activates caspase-1, led to the maturation of IL-1β in Gaucher macrophages. We show that inflammasome activation in these cells is the result of impaired autophagy. Treatment with the small-molecule glucocerebrosidase chaperone NCGC758 reversed these defects, inducing autophagy and reducing IL-1β secretion, confirming the role of the deficiency of lysosomal glucocerebrosidase in these processes. We found that in Gaucher macrophages elevated levels of the autophagic adaptor p62 prevented the delivery of inflammasomes to autophagosomes. This increase in p62 led to activation of p65-NF-kB in the nucleus, promoting the expression of inflammatory cytokines and the secretion of IL-1β. This newly elucidated mechanism ties lysosomal dysfunction to inflammasome activation, and may contribute to the massive organomegaly, bone involvement and increased susceptibility to certain malignancies seen in Gaucher disease. Moreover, this link between lysosomal storage, impaired autophagy, and inflammation may have implications relevant to both Parkinson disease and the aging process. Defects in these basic cellular processes may also provide new therapeutic targets.

Keywords: Gaucher disease; autophagy; glucocerebrosidase; inflammasome; interleukin-1β; lysosome; macrophage.

Figure 1

Gaucher macrophages show inflammasome activation and secrete IL‐1β (A) Control (C) and N370S/N370S (P) peripheral blood monocytes were treated with lipopolysaccharide (LPS) or LPS and ATP and the supernatant probed for IL‐1β. (B) An ELISA for activated IL‐1β was performed using the supernatants from patient and control macrophages treated with LPS. All samples were evaluated in duplicate by ELISA. n = number of patients. (C) Macrophage lysates were probed for IL‐6. Statistical significance; P < 0.05(*), P < 0.01(**), P < 0.001(***).

Figure 2

Caspase‐1 activation and increased ubiquitinated protein apoptosis‐associated speck‐like proteins (ASC) in Gaucher macrophages (GMs). (A) ELISA or (B) Total supernatants or total protein lysates from treated macrophages from patients (P) and controls (C) were probed for IL‐1β. (C) Control (C) and GMs (P) were immuno‐stained for LC3 (red) and NLRP3 (green) after treatment with lipopolysaccharide (LPS) (100 ng) and ATP (5 mm) for 1 h. Cells were imaged using a confocal microscope (Z‐stack with 0.5 μm thickness). Co‐localization was evaluated using Imaris software. Merged channels are shown as yellow. Pictures represent seven independent experiments performed on cells from seven different patients. Single channels are presented in the supplements. (D) Caspase‐1 was analyzed in total lysate and supernatant by immunoblotting, after treatment with LPS alone or LPS (100 ng)+ATP (5 mm) or rapamycin (25 nm). Data represent five independent experiments. (E) Total protein from control and Gaucher macrophages was immunoblotted for ASC after treatment with LPS (100 ng) and ATP (5 mm), and then rapamycin (25 nm), 3MA (5 mm) or Baf.1A (10 μm) for 40 min. ASC expression was normalized to β‐actin. The blot is representative of seven independent experiments performed on samples from seven different patients and controls. P < 0.05(*) and P < 0.01(**), P < 0.001(***). (F) Control and GMs were immunostained for p62 (red) and ASC (green) in the absence and presence of LPS (100 ng) and ATP (5 mm) and then imaged by confocal microscopy. Merged channel volumes are shown in yellow, and insets showing surface renderings of three‐dimensional reconstruction using Imaris software are shown in the far right panels.

Figure 3

Impaired autophagy results in activation of inflammasomes in Gaucher macrophages (GMs). (A) Control (C) and GMs (P) (n = 4) were treated with lipopolysaccharide (LPS) (100 ng) or LPS+ATP (5 mm). 10 μm Baf.1A was added for 40 min after stimulation with LPS + ATP. Total lysates were run on SDS‐PAGE gels, and blots were probed with antibodies to VP34, Atg7, p62, LC3, Atg 16L1, and β‐actin. (B) LC3II protein levels normalized to β‐actin. (C) Results of a puncta assay performed in control and GMs in four independent experiments. Graph reflects the number of LC3 puncta counted per cell. Control and GMs, treated as described above, were then immunostained for (D) LC3 (red) and IL‐1β (green). (E) p62 (red) and apoptosis‐associated speck‐like proteins (green), (F) NLRP3 (green) and LC3 (red) and (G) IL‐1β (green) and LC3 (red). Images represent 20 pictures taken in 5 independent experiments (63X magnification and scale bar; 5μ).

Figure 4

Treatment of Gaucher macrophages (GMs) macrophages with the small‐molecule NCGC758 results in reduced IL‐1β secretion (A) Control (C) and GMs (P) were treated with NCGC758 (8 μm) in the presence and absence of Gaucher erythrocyte ghosts. Total lysates were immunoblotted and probed for LC3 and p62. (B) Gaucher cells were treated with NCGC758 or imiglucerase (20 μm) followed by LPS+ATP (5 mm). Total protein lysates were run on SDS‐PAGE and were probed for LC3, P62, and Atg16L1. Blots represent two independent experiments. (C,D) Gaucher macrophages treated with NCGC758 in the presence of erythrocyte ghosts, or treated with LPS and ATP, were immunostained for (C) LC3 (green) and Lamp2 (red) and (D) LC3 (red) and p62 (green). Z‐stack images were acquired using a Zeiss 510 confocal microscope (63× magnification). Insets corresponding to the regions marked show higher magnifications of the areas outlined with the position of the xy‐, xz‐, and yz‐slices that are shown for each of the 3D stacks. Images are taken at the same laser settings and are representative of three independent experiments (scale bar; 5μ). (E,F) Control (C) and GMs (P) were treated with NCGC758 (8 μm) or Imiglucerase in the presence and absence of LPS+ATP and the supernatants (E) or total protein lysates (F) were probed for IL‐1β. Blots represent two independent experiments. n = number of patients.

Figure 5

p65‐NF‐kB activation in Gaucher macrophages (GMs). (A) Cytosolic and nuclear fractions from GMs (P) and control (C) macrophages were immunoblotted for p65‐NF‐kB (NFKB) after treatment with lipopolysaccharide (LPS) (100 ng) and supplementation with 3MA (5 mm) and/or rapamycin (25 nm). GAPDH and histone were used as the standards for the cytosolic and nuclear fractions, respectively. Normalized expression levels are shown in the graphs, representing four independent experiments. (B) Nuclear and cytosolic fraction from control (C) and GMs (P), before and after LPS, ATP, and 3MA treatment, were probed for p62. Data represent three independent experiments. (C) Control (C) and GMs (P) were treated with LPS (100 ng) and ATP (5 mm) in the presence and absence of Baf.1A (10 μm) and were stained for p65‐NF‐kB (red) and p62 (green). Images represent 30 pictures taken in 6 independent experiments (63× magnification, scale bar; 5μ). Insets show higher magnification of areas outlined (a, b). (D) Control and GMs treated with LPS (100 ng) alone and supplemented with rapamycin (25 nm) or Baf.1A (10 μm), were stained for p65‐NF‐kB (green) and Lamp2 (red). Images represent 15 pictures from four independent experiments. Insets illustrate co‐localization of NF‐kB with DAPI, a nuclear marker.

Figure 6

Inflammasome activation due to impaired autophagy in Gaucher macrophages (GMs) (1) In both control and GMs, lipopolysaccharide (LPS) priming induces activation of p65‐NF‐kB, which is translocated to the nucleus, leading to production of pro‐IL‐1β in the cytosol. In control macrophages (1a), LPS stimulates accumulation of ubiquitinated p65‐NF‐kB, which is further recognized by p62, delivered to autophagosomes, and degraded in the lysosome (1a, solid line). In GMs (1b, dashed line), impaired autophagy prevents degradation of p65‐ NF‐kB through autophagy and results in its activation (1b). (2) In both control and GMs, stimulation by both LPS and extracellular ATP leads to inflammasome complex formation (NLRP3, apoptosis‐associated speck‐like proteins (ASC) and caspase1) and activation. (3) Activated inflammasomes undergo ubiquitination of ASC, leading to p62‐mediated engulfment of inflammasomes by autophagosomes. Pro‐IL‐1β conversion to active IL‐1β is limited due to the destruction of activated inflammasomes by autophagolysosomes. In GMs, defective autophagy and lysosomal dysfunction inhibit the elimination of active inflammasomes through autophagy, resulting in the upregulation and secretion of IL‐1β (3).