Activation of inflammasomes requires intracellular redistribution of the apoptotic speck-like protein containing a caspase recruitment domain

Abstract

Activation of caspase 1 is essential for the maturation and release of IL-1beta and IL-18 and occurs in multiprotein complexes, referred to as inflammasomes. The apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is the essential adaptor protein for recruiting pro-caspase 1 into inflammasomes, and consistently gene ablation of ASC abolishes caspase 1 activation and secretion of IL-1beta and IL-18. However, distribution of endogenous ASC has not yet been examined in detail. In the present study, we demonstrated that ASC localized primarily to the nucleus in resting human monocytes/macrophages. Upon pathogen infection, ASC rapidly redistributed to the cytosol, followed by assembly of perinuclear aggregates, containing several inflammasome components, including caspase 1 and Nod-like receptors. Prevention of ASC cytosolic redistribution completely abolished pathogen-induced inflammasome activity, which affirmed that cytosolic localization of ASC is essential for inflammasome function. Thus, our study characterized a novel mechanism of inflammasome regulation in host defense.

Figure 1. Nuclear localization of ASC

(A) HEK293 cells were transiently transfected with myc-ASC, fixed, and immunostained with anti-myc and Alexa Fluor 488-conjugated antibodies, while GFP-ASC expressing THP-1 cells were fixed and cytospun onto glass slides. ASC either localized diffusively throughout the cell (upper panel) or into a perinuclear aggregate (middle panel), while stable expression of GFP-ASC in THP-1 cells showed nuclear localization (lower panel). (B) Subcellular localization of endogenous ASC was examined in primary human monocytes and several monocytic cell lines. Cells were fixed and ASC was immunostained with monoclonal antibodies from MBL (monocytes) (1^st panel), and polyclonal antibodies from Chemicon, Calbiochem, and custom raised (HL-60, U-937, THP-1) (2^nd, 3^rd, and 4^th panel). THP-1 cells were also immunostained in the presence of a 1000x molar excess of the ASC-specific peptide that is recognized by the CS3 anti-ASC antibody (5^th panel). Secondary Alexa Fluor 488-conjugated antibodies were used in combination with ToPro-3 and Alexa Fluor 546-conjugated phalloidin to counter stain nuclei (DNA) and the actin cytoskeleton to outline cells, respectively. Images were acquired by laser scanning confocal microscopy, and the panels from left to right are showing ASC (green), nucleus (blue), actin (red), and a merged image. (C) THP-1 lysates (75 μg) were analyzed by immunoblot using the anti-ASC antibodies used in immunofluorescence microscopy. An asterisk denotes a splice form of ASC, which is not recognized by our custom-raised polyclonal anti-ASC antibody (CS3). (D) HEK293T cells were either mock transfected or transfected with a myc-tagged ASC expression construct. Cleared cell lysates were analyzed by immunoblot using the anti-ASC antibodies used in immunofluorescence microscopy and anti-myc antibodies (Santa Cruz Biotech) as control. Blots were also stripped and re-probed for GAPDH to demonstrate equal loading.

Figure 2. Cytosolic redistribution of ASC in response to inflammatory stimulation of monocytes

(A) Subcellular localization of ASC was analyzed by immunofluorescence in primary monocytes and THP-1 monocytes following treatment with E. coli total RNA (2 μg/ml) for 30 minutes, using the monoclonal anti-ASC antibody described in Fig. 1. Images were acquired by laser scanning confocal microscopy. Panels from left to right are showing ASC (green), nucleus (blue), actin (red), and a merged image. (B) Subcellular localization of ASC was determined by subcellular fractionation of control and E. coli total RNA (2 μg/ml)-activated THP-1 cells. 10⁶ cells were fractionated by differential centrifugation into nuclear (N) and cytosolic (C) fractions, and protein lysates (50 μg) were analyzed by immunoblotting with anti-ASC and HRP-conjugated secondary antibodies. Fractionation efficiency was controlled by re-probing membranes with anti-GAPDH (cytosolic) and anti-Lamin A (nuclear) antibodies. *denotes two cross reactive proteins in the cytosolic fraction following E. coli total RNA treatment.

Figure 3. Aggregate formation of endogenous ASC in response to inflammatory stimulation in macrophages

Subcellular localization of ASC was analyzed by immunofluorescence in primary macrophages (A) and PMA-differentiated THP-1 macrophages (B,C), using mono- and polyclonal anti-ASC and Alexa Fluor 488-conjugated secondary antibodies in combination with ToPro-3 nuclear stain and Alexa Fluor 546 conjugated phalloidin to visualize actin. Images were acquired by laser scanning confocal microscopy. All panels show ASC (green), nucleus (blue), actin (red), and a merged image from left to right. (A) Primary macrophages, either untreated (upper panel) or E. coli total RNA treated (2 μg/ml) (lower panel), were immunostained using the CS3 polyclonal anti-ASC antibody. (B) Untreated (upper panel) and E. coli total RNA (2 μg/ml) treated THP-1 macrophages (middle and lower panels) were immunostained using the polyclonal anti-ASC antibody in the absence (middle panel) or in the presence (lower panel) of a 1000x molar excess of the ASC-specific peptide (note the loss of specific ASC aggregate staining in the panel with peptide competition). (C) THP-1 macrophages were activated with 2×10⁵ cfu/ml heat killed Staphylococcus aureus (HKSA) (upper panel) and Legionella pneumophilia (HKLP) (lower panel), and immunostained with the CS3 polyclonal antibody.

Figure 4. Aggregation of ASC occurs within one hour of inflammatory stimulation of macrophages and depends on the continued presence of the stimulus

Subcellular localization of ASC in THP-1 macrophages was analyzed by immunofluorescence following stimulation of cells with E. coli total RNA (2 μg/ml) for the indicated times. Cells were fixed at 0 minutes, 30 minutes, 1 hour, 6 hours, and 24 hours post-stimulation. In addition, after 6 hours of stimulation, cells were washed extensively and cultured for additional 12 hours in the absence of E. coli total RNA. Fixed cells were stained with the polyclonal CS3 anti-ASC, and Alexa Fluor 488-conjugated secondary antibodies. DNA and the actin cytoskeleton were visualized as above. The panels from left to right are showing ASC (green), nucleus (blue), actin (red), and a merged image.

Figure 5. Caspase-1 and NLRP3 co-localize with ASC in aggregates

All images show PMA-differentiated THP-1 macrophages, except (E), which are HEK293T cells. (A) Cells were treated with E. coli total RNA (2 μg/ml) and immunostained with polyclonal anti-ASC and Alexa Fluor 546-conjugated antibodies (red), monoclonal anti-β-tubulin and Alexa Fluor 488-conjugated antibodies (green) and the nuclear stain DAPI (blue). The insert in the lower right half highlights the ring-shaped ASC-containing aggregate. The scale bar measures 10 μm and 1 μm, respectively. (B) E. coli total RNA (2 μg/ml)-treated cells were immunostained with polyclonal anti-ASC and Alexa Fluor 546-conjugated antibodies (red) and DAPI (blue). 20 optical sections at 0.6 μm were captured, deconvoluted and assembled into a 3D structure, showing xy, yz, and xz views of the aggregate. The scale bar is 10 μm. (C, D) Untreated (upper panel) or E. coli total RNA (2 μg/ml)-treated cells (lower panel) were fixed and immunostained with (C) polyclonal anti-caspase-1 and monoclonal β-tubulin and Alexa Fluor 546 and 488-conjugated secondary antibodies, respectively. (D) Polyclonal anti-ASC and monoclonal anti-caspase-1 antibodies and Alexa Fluor 488- and 546-conjugated secondary antibodies, respectively. DNA was visualized with DAPI, and panels from left to right show (C) caspase-1 (red), nucleus (blue), β-tubulin (green), and a merged image and (D) ASC (green), caspase-1 (red), nucleus (blue), and a merged image. (E) Flag-tagged ASC, HA-tagged caspase-1 and myc-tagged NLRP3^R260W were transiently transfected into HEK293 cells and immunostained with rabbit anti-ASC, mouse anti-NLRP3, and rat anti-HA antibodies and Alexa Fluor 546-, 647-, and 488-conjugated secondary antibodies, respectively, to determine co-localization of all three proteins in transfected cells. Panels from left to right show ASC (red), NLRP3^R260W (blue), caspases-1 (green), phase, and a merged image. (F) E. coli total RNA (2 μg/ml)-treated THP-1 macrophages were immunostained with rabbit anti-ASC and goat anti-NLRP3 antibodies and Alexa Fluor 488- and 546-conjugated secondary antibodies, respectively. Panels from left to right show ASC (red) and NLRP3 (green) and a merged image.

Figure 6. ASC aggregate formation is linked to the maturation of IL-1β

(A) Normalized culture supernatants from resting and E. coli RNA (2 μg/ml)-activated primary human macrophages were analyzed by ELISA for released IL-1β. Results represent an average of two independent experiments, and are presented as fold release compared to untreated macrophages. (B) Culture supernatants from THP-1 cells stably transfected with either an shRNA targeting ASC (black bars) or a control shRNA targeting luciferase (gray bars) and left either untreated, or treated with E. coli RNA (2 μg/ml), HKLP (2×10⁵ cfu/ml) or HKSA (2x10⁵ cfu/ml) were analyzed as above for released IL-1β. Cells were previously FACS sorted for GFP expression, which is encoded from the pRNATin-H1.4 (Genscript) shRNA vector backbone. Results represent an average of three independent experiments, and are presented as pg/ml of released IL-1β. The insert shows an immunoblot of control shRNA and ASC shRNA expressing THP-1 cells for ASC and GAPDH as a loading control. (C) ASC-containing aggregates were analyzed following treatment with E. coli total RNA (2 μg/ml) for 6 hours by immunofluorescence in a mixed population of shRNA ASC transfected cells before FACS sorting for GFP expression of the ASC shRNA. ASC was immunostained with the polyclonal CS3 antibody, and images were acquired by laser scanning confocal microscopy. Panel shows from left to right ASC (red), ASC shRNA (green), nucleus (blue), and a merged image. Note that the cell on the left side (white arrow) encodes the ASC shRNA, as indicated by the GFP positive signal and therefore does not form ASC-containing aggregates, while the cell on the right is GFP negative, thus does not encode the ASC shRNA and therefore shows aggregated ASC.

Figure 7. ASC localization to the cytosol is required for inflammasome formation and efficient IL-1β secretion

(A) Untreated and E. coli RNA-treated THP-1 macrophages were immunostained with anti-IL-1β and Alexa Fluor 488-conjugated secondary antibodies. Nuclei and actin were visualized as above. Panels show from left to right IL-1β (green), nucleus (blue), actin (red), and a merged image. (B) Myc-tagged ASC (upper panel) and myc-tagged NLS-ASC (lower panel) were transiently transfected into HEK293 cells and immunostained with monoclonal anti-myc and Alexa Fluor 546-conjugated secondary antibodies. Nuclei and actin were visualized as above. Panels show from left to right ASC (red), actin (green), nucleus (blue), and a merged image. Images in (A, B) were acquired by laser scanning confocal microscopy. (C) Inflammasomes consisting of a constitutively active NLRP3^R260W, pro-IL-1β, pro-caspase-1, and either ASC or NLS-ASC, were transiently reconstituted in HEK293 cells, as indicated, and inflammasome activity was assayed by analyzing secreted IL-1β by ELISA. Results represent an average of at least three independent experiments +/− SD. Cleared and normalized cellular lysates were analyzed by immunoblot for expression of all transfected inflammasome components, as indicated. (D) THP-1 cells were stably infected with a VSV-G pseudo-typed recombinant retrovirus encoding either red fluorescent protein (RFP)-fused ASC or RFP-fused NLS-ASC with expression levels comparable to endogenous ASC. Mock-infected, RFP-ASC, and RFP-NLS-ASC expressing cells were transiently transfected with two pooled ASC-specific siRNAs to knock-down expression of endogenous ASC, but not RFP-ASC or RFP-NLS-ASC. Cells were seeded into fresh wells 24 hours post nucleofection, and where indicated treated with LPS and E. coli RNA for 12 hours. Secreted IL-1β was determined in culture supernatants by ELISA and results are presented as pg/ml of 10⁶ cells, and represent an average of three independent experiments +/− SD. The insert shows stable expression of RFP-ASC and RFP-NLS-ASC compared to endogenous ASC in THP-1 cells (upper panel) and the siRNA-mediated reduction of endogenous ASC (lower panel) by immunoblot. An asterisk denotes expression of ASC and NLS-ASC containing silent point mutations in the sequence recognized by the siRNA preventing its degradation.

Figure 8. A model for inflammasome formation and activation

Cross talk between the TLR and NLR system has been proposed, where initial DAMP recognition by TLRs triggers transcriptional up-regulation of pro-IL-1β and other inflammasome components. Subsequently, DAMP recognition by NLRs is required for maturation of pro-IL-1β and pro-IL-18. Our data indicate that DAMP recognition also causes redistribution of ASC from the nucleus to the cytosol by a yet elusive mechanism, which then can be recruited to activated NLRs to assemble inflammasomes. The perinuclear aggregates in activated macrophages contain the core inflammasome proteins and might represent inflammasomes.