Nanobodies dismantle post-pyroptotic ASC specks and counteract inflammation in vivo

Abstract

Inflammasomes sense intracellular clues of infection, damage, or metabolic imbalances. Activated inflammasome sensors polymerize the adaptor ASC into micron-sized "specks" to maximize caspase-1 activation and the maturation of IL-1 cytokines. Caspase-1 also drives pyroptosis, a lytic cell death characterized by leakage of intracellular content to the extracellular space. ASC specks are released among cytosolic content, and accumulate in tissues of patients with chronic inflammation. However, if extracellular ASC specks contribute to disease, or are merely inert remnants of cell death remains unknown. Here, we show that camelid-derived nanobodies against ASC (VHH_ASC ) target and disassemble post-pyroptotic inflammasomes, neutralizing their prionoid, and inflammatory functions. Notably, pyroptosis-driven membrane perforation and exposure of ASC specks to the extracellular environment allowed VHH_ASC to target inflammasomes while preserving pre-pyroptotic IL-1β release, essential to host defense. Systemically administrated mouse-specific VHH_ASC attenuated inflammation and clinical gout, and antigen-induced arthritis disease. Hence, VHH_ASC neutralized post-pyroptotic inflammasomes revealing a previously unappreciated role for these complexes in disease. VHH_ASC are the first biologicals that disassemble pre-formed inflammasomes while preserving their functions in host defense.

Keywords: arthritis; extracellular inflammasomes; gout; nanobodies; pyroptosis.

Figure 1. VHH_ASC blocks the extracellular pro‐inflammatory function of ASC specks

1. Mouse IL‐1β (mIL‐1β) concentrations in cell‐free supernatants of LPS‐primed BMDMs (200 ng ml⁻¹, for 2 h), that were stimulated with in vitro‐generated human ASC specks (200 µg ml⁻¹, O/N). ASC specks were pre‐incubated with 200 µg ml⁻¹ of an anti‐human ASC nanobody (VHH_ASC), its mutated variant, mutVHH_ASC or 50 µg ml⁻¹ of polyclonal ASC antibody (a‐ASC pAb, AL177) for 15 min at RT. Data is pooled from at least two independent experiments, each represented by a different symbol, and is displayed as floating bars with the max/min values and mean (thicker band).

2. Immunoblot analysis of pro‐ (p31) and cleaved (p17) IL‐1β, and pro‐ (p45) and cleaved (p20) caspase‐1 in cytosolic fractions of ASC deficient (Pycard ^−/−) immortalized murine macrophages that were primed with LPS (200 ng ml⁻¹, for 3 h). Fractions were incubated with recombinant ASC specks in the presence of 2, 20 or 200 µg ml⁻¹ of VHH_ASC or mutVHH_ASC. One representative of three independent experiments is shown.

3. Quantitative analysis of band densitometry of three independent experiments as shown in B. Data represents the fold change from all conditions (lanes 1–9) vs. Pycard ^−/− lysates incubated with ASC specks alone (lane 2). ^ns P > 0.05; *P < 0.05; **P < 0.005; ***P < 0.0002, One‐way ANOVA, multiple comparison (Dunnet test). Data is displayed as floating bars with the max/min values and mean (thicker band).

Source data are available online for this figure.

Figure EV1. Binding activity and species specificity of ASC VHHs

Lysates of HEK 293T cells transiently expressing HA‐tagged VHH_ASC (wild‐type or point mutants), an unrelated control (VHH_NP1) and the indicated bait proteins fused to Renilla luciferase were used to immunoprecipitate VHHs with immobilized anti‐HA antibody. Renilla luciferase activity of the co‐immunoprecipitated proteins was measured and normalized to the input luciferase. Data represents mean values ± SEM from three independent experiments. ****P < 0.0001, One‐way ANOVA, multiple comparison (Tukey test). 'TYD‐KKK' indicates the triple mutant T57K, Y59K, and D62K.

Figure 2. VHH_ASC disrupts the “prionoid” activities and disassembles ASC specks

1. Time‐lapse confocal imaging of the in vitro nucleation of soluble ASC‐mTurquoise (red) by ASC‐TagGFP (ASC‐GFP) specks (green), that were left untreated (None), or pre‐incubated with VHH_ASC or mutVHH_ASC (200 µg ml⁻¹ for 15 min). Scale bars: 10 μm.

2. Median fluorescence intensity (MFI) of mTurquoise graphed over time showing its polymerization seeded by ASC‐TagGFP specks. Each line shows the mTurquoise MFI around a seeding ASC‐TagGFP speck. Data is one representative of three independent experiments.

3. WES capillary electrophoresis and immunoblotting of DSS cross‐linked in‐vitro‐generated human ASC‐mTurquoise (ASC) specks that were incubated for 1 h (RT) with 2, 20 or 200 µg ml⁻¹ of VHH_ASC, mutVHH_ASC or with 20 µg ml⁻¹ of a polyclonal anti‐ASC Ab (a‐ASC pAb). Data is from one representative of four independent experiments.

Source data are available online for this figure.

Figure 3. VHH_ASC and VHH_mASC prevent IL‐1β release and ASC speck formation induced by PFO, but not nigericin in primary human and mouse cells

1. A–D

   (A, B) Human IL‐1β (hIL‐1β) concentrations in cell‐free supernatants of LPS‐primed (10 ng ml⁻¹, 150 min) primary human macrophages that were left untreated, or pre‐incubated with VHH_ASC or mutVHH_ASC (100 µg ml⁻¹), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before stimulation with (A) nigericin (10 µM), or (B) PFO (30 ng ml⁻¹) for 2 h. (C‐D) Mouse IL‐1β (mIL‐1β) concentrations in cell‐free supernatants of LPS‐primed mouse BMDMs (200 ng ml⁻¹, 150 min), incubated with VHHs, CRID3 or VX765, before activation with nigericin (10 µM), or PFO (250 ng ml⁻¹). Data is combined from two independent experiments, each performed with two donors (A, B) or mice (C, D), represented with individual symbols (4 donors or mice in total). Data is displayed as floating bars with the max/min values and mean (thicker band).

2. E, F

   (E) Epifluorescence microscopy imaging and (F) quantification of ASC speck formation in BMDMs from ASC‐mCitrine (Green) transgenic mice. Cells were primed with LPS (200 ng ml⁻¹, 150 min), pre‐treated with VX‐765 (50 µM, 30 min), then treated with VHH_ASC, VHH_mASC (100 µg ml⁻¹) or CRID3 (50 µM) for another 30 min before stimulation with nigericin (top), or PFO (bottom) for 2 h and finally fixed with 4% PFA. Nuclei was stained with DRAQ5 (Blue). Scale bars: 100 μm. Images in (E) are from one representative out of three independent experiments that were quantified in F. Data in F is displayed as floating bars with the max/min values and mean (thicker band).

Data information: **P < 0.005; ***P < 0.0002; ****P < 0.0001, One‐way ANOVA, multiple comparison (Tukey test).

Figure EV2. VHH_ASC preserves endogenous inflammasome activation in human living cells

1. A, B

   Cell viability (CTB) assay on LPS‐primed primary human macrophages that were left untreated, or pre‐incubated with VHH_ASC or mutVHH_ASC (100 µg ml⁻¹), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being activated with (A) nigericin (10 µM), or (B) PFO (30 ng ml⁻¹) for 2 h. Data is from the experiments displayed in Fig 3A and B.

2. C

   Human IL‐1β (hIL‐1β) concentrations in cell‐free supernatants (left), and cell viability assay (right) of LPS‐primed primary human macrophages that were incubated with VHH_ASC or mutVHH_ASC (100 µg ml⁻¹), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being stimulated with 2.5 mM ATP.

3. D

   hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of PMA‐differentiated THP‐1 cells treated with VHH_ASC or mutVHH_ASC (100 µg ml⁻¹), CRID3 (10 µM) or VX‐765 (50 µM) for 30 min before 4.5 h stimulation with 250 µg ml⁻¹ MSU crystals.

4. E

   hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of LPS‐primed primary human macrophages that were incubated with VHH_ASC or mutVHH_ASC (100 µg ml⁻¹), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being stimulated with 0.1 µg ml⁻¹/0.5 µg ml⁻¹ mixture of LFn‐BsaK and PA for 2 h.

5. F

   hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of Pam3CysK4‐primed (1 µg ml⁻¹) primary human CD14⁺ monocytes that were incubated with VHH_ASC or mutVHH_ASC (100 µg ml⁻¹), or VX‐765 (50 µM) for 30 min before being stimulated with 1 µg ml⁻¹ TcdA.

6. G

   hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of keratinocyte cells (N‐TERT) that were treated with VHH_ASC or mutVHH_ASC (100 µg ml⁻¹), or VX‐765 (50 µM), then directly stimulated with 30 µM Val‐boroPro (VbP) for 22 h.

7. H

   hIL‐1β concentrations in cell‐free supernatants (top), and cell viability assay (bottom) of PMA‐differentiated THP‐1 cells treated with IFNγ (500 U ml⁻¹) for 16 h and that were incubated with VHH_ASC or mutVHH_ASC (100 µg ml⁻¹), CRID3 (10 µM) or VX‐765 (50 µM) for 30 min before 2 h stimulation with 1 µg ml⁻¹ poly(dA:dT) in complex with Lipofectamine 2000.

Data information: Data is representative of either two independent experiments, each run with one to two donors (A–C, E, F, 3 or 4 donors in total) or at least 3 independent experiments (D, G, H). Each symbol represents one donor or independent experiment. ^ns P > 0.05; *P < 0.05; **P < 0.005; ***P < 0.0002; ****P < 0.0001, One‐way ANOVA, multiple comparison (Tukey test). Data is displayed as floating bars with the max/min values and mean (thicker band).

Figure EV3. VHH_mASC is specific to ASC^PYD but retains the same activity as VHH_ASC

1. A

   Specificity of different single‐domain antibodies (VHHs) probed by ELISA. Recombinant murine ASC as an eGFP fusion or eGFP alone (GFP‐LPETG) as control, were coated onto ELISA plates at 1 µg ml⁻¹/well. Wells were incubated with HA‐tagged VHHs (100 nM), anti‐HA‐tag mouse mAb coupled to HRP, and the HRP substrate TMB. Binding was quantified by measuring the absorbance at 450 nm.

2. B, C

   Lysates of HEK 293T cells transiently expressing HA‐tagged VHH_mASC or VHH_ASC and the indicated bait proteins fused to Renilla luciferase were used to immunoprecipitate VHHs with immobilized anti‐HA antibody. Renilla luciferase activity of the co‐immunoprecipitated proteins was measured and normalized to the input luciferase. Data represents mean values ± SEM from three independent experiments.

3. D, E

   Cell viability (CTB) assay on LPS‐primed (200 ng ml⁻¹) primary mouse BMDMs that were left untreated, or pre‐incubated with VHH_ASC or VHH_mASC (100 µg ml⁻¹), CRID3 (50 µM) or VX‐765 (50 µM) for 30 min before being activated with (D) nigericin (10 µM), or (E) PFO (250 ng ml⁻¹) for 2 h. Data is from the experiments displayed in Fig 3C and D. Data is displayed as floating bars with the max/min values and mean (thicker band).

4. F

   Mouse IL‐1β (mIL‐1β) concentrations in cell‐free supernatants (left), and cell viability assay (right) of LPS‐primed mouse BMDMs incubated with VHHs, CRID3 or VX–765, before stimulation with 0.1 µg ml⁻¹/0.5 µg ml⁻¹ mixture of LFn‐BsaK and PA for 2 h. Each symbol represents one mouse. Data is displayed as floating bars with the max/min values and mean (thicker band).

Data information: ^ns P > 0.05; ***P < 0.001; ****P < 0.0001, Two‐way (A, C) or One‐way (B, D–F) ANOVA, multiple comparison (Tukey test).

Figure 4. Contribution of GSDMD in the effect of VHH_ASC on the release of IL‐1β and cell death induced by PFO or nigericin

THP‐1 cells expressing a Dox‐inducible CRISPR‐Cas9 cassette targeting GSDMD were left untreated (–), or treated with 1 µg ml⁻¹ Dox for one or two cycles of 72 h (1×, or 2× respectively).

1. Immunoblot analysis of GSDMD expression following the indicated course of Dox treatment and PMA‐differentiation, as indicated. Data is from one representative of two independent experiments.

2. IL‐1β concentration or percentage of LDH released into cell‐free supernatants of PMA‐differentiated THP‐1 cells that were treated with VHH_ASC (200 µg ml⁻¹) or CRID3 (25 µM) for 30 min prior to stimulation with nigericin (10 µM, left panels) or PFO (30 ng ml⁻¹, right panels) for 2 h. Data is average of experimental duplicates from three independent experiments, each represented by a different symbol. ^ns P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, Two‐way ANOVA, multiple comparison (Tukey test). Data is displayed as floating bars with the max/min values and mean (thicker band).

3. Live confocal imaging of PMA‐differentiated and nigericin‐treated (10 µM) THP‐1 cells expressing human ASC‐GFP (green) in the presence of AlexaFluor647‐labeled VHH_ASC (VHH_ASC‐AF647, 10 µg ml⁻¹, cyan) in the medium. Cells were either left untreated (–) or incubated with VX‐765 (50 µM) for 1 h prior to nigericin stimulation. Nuclei were stained with Hoechst 34580 (magenta). Scale bar: 50 µm. Data is from one representative out of two independent experiments.

Source data are available online for this figure.

Figure EV4. Contribution of GSDMD in the effect of VHHASC on the internalization of VHH_ASC in cells stimulated with PFO or nigericin

THP‐1 cells expressing a Dox‐inducible CRISPR‐Cas9 cassette targeting GSDMD gene were left untreated (–) or treated with 1 µg ml⁻¹ Dox for one or two cycles of 72 h (1×, or 2× respectively).

1. A, B

   Confocal microscopy images of GSDMD competent (–Dox) or GSDMD‐KO (+Dox) cells primed with PMA and that were either left untreated or treated with (A) nigericin (10 µM) or (B) PFO (30 ng ml⁻¹) for the indicated periods of time and in the presence of AlexaFluor647‐labeled VHH_ASC (VHH_ASC‐AF647, 10 µg ml⁻¹, blue) and propidium iodide (PI, 3.33 µg ml⁻¹, red). White arrows indicate of cells with intracellular VHH_ASC‐AF647 signal. Dashed circles define boundaries of representative cells. Data is from one representative out of three independent experiments. Scale bar: 10 µm. Also see Movie EV1.

Figure 5. Pyroptosis allows VHH_ASC to access the cytosol and target ASC specks

1. A

   Wide‐field fluorescence microscopy imaging of live PMA‐differentiated and nigericin‐ (10 µM, top) or PFO‐activated (25 ng ml⁻¹, bottom) THP‐1 expressing human ASC‐GFP. Cells were incubated with VHH_ASC (200 µg ml⁻¹) alone or in the presence of the caspase‐1 inhibitor VX‐765 (50 µM). Nuclei were stained with DRAQ5 (blue). Cells were imaged live with a CellDiscoverer 7 microscope. For each condition, a total of 8 positions within 2 wells (2 × 4 images/well) were imaged. Data is maximal intensity projections from Z‐stacks. Scale bars: 50 µm. See also Movie EV2.

2. B

   Graphic representation of the percent of specking cells over time. Maximal intensity projections from Z‐stacks were generated for each image set before the number of cells and specks per field were calculated using CellProfiler. (A) represents images from one experiment out of three independent experiments of which percentage of specking cells data is represented in (B) with each line representing one field‐of‐view.

3. C, D

   (C) Flow cytometry quantification, and (D) immunoblotting analysis of ASC‐GFP oligomers (DSS cross‐linked) in extracellular specks recovered from supernatants of cells treated as in A. Data in (C) is from two independent experiments (each represented by a different symbol). And in (D) is from one out of at least two independent experiments. Data is displayed as floating bars with the max/min values and mean (thicker band).

4. E

   Mouse BMDMs expressing ASC‐mCitrin and primed with LPS (200 ng ml⁻¹) for 3 h were pre‐treated with VHH_mASC (200 µg ml⁻¹) alone or in the presence of the caspase‐1 inhibitor VX‐765 (50 µM) for 30 min. Cells were then treated with a mixture of LFn‐MxiH and PA (Mx/PA, 100 ng ml⁻¹/1 µg ml⁻¹) or PFO (250 ng ml⁻¹) and imaged as in (A) for 3 h. Data is a graphic representation of specking cells over time calculated as in (B). Data is from one experiment out of three independent experiments. See also Movie EV6.

Source data are available online for this figure.

Figure EV5. VHH_ASC disrupts post‐pyroptotic ASC specks

1. PMA‐differentiated (50 ng ml⁻¹) THP‐1 ASC‐GFP cells were treated with IFNγ (500 U ml⁻¹) for 16 h. Then, cells were pre‐incubated with VHH_ASC (100 µg ml⁻¹), or VX‐765 (50 µM) for 30 min before being activated with poly(dA:dT) (dA:dT, 1 µg ml⁻¹) and imaged live with a CellDiscoverer 7 microscope as described in Fig 4 over 3 h. Data is a graphic representation of the percent of specking cells over time. Maximal intensity projections from Z‐stacks were generated for each image set (8 images per condition) before the number of cells and specks per field were calculated using CellProfiler. Also see Movie EV3.

2. Flow cytometric quantification (left), or western‐blot (right, following DSS cross‐linking) assessement of GFP⁺ specks in the supernatants of cells pre‐treated with VHH_ASC or VX‐765 and then activated with poly(dA:dT) for 3.5 h. Data in left panel is displayed as floating bars with the max/min values and mean (thicker band).

3. Flow cytometric quantification or western‐blotting of GFP+ specks released by PMA‐differentiated THP‐1 ASC‐GFP cells pre‐incubated with VHH_ASC (100 µg ml⁻¹), or VX‐765 (50 µM) for 30 min before stimulation with LFn‐MxiH/PA (100 ng ml⁻¹/1 µg ml⁻¹) for 3 h and imaged live with a CellDiscoverer 7 microscope. Also see Movie EV4.

4. GFP⁺ specks detected by flow cytometry (left) or western‐blot (right, following DSS cross‐linking) in the supernatants of cells pre‐treated with VHH_ASC or VX‐765 and then activated with LFn‐MxiH/PA for 3.5 h. Data in left panel is displayed as floating bars with the max/min values and mean (thicker band).

5. PMA‐differentiated THP‐1 ASC‐GFP cells were pre‐incubated with VHH_ASC (100 µg ml⁻¹), or VX‐765 (50 µM) for 30 min before stimulation with MSU crystals (250 µg ml⁻¹) for 4.5 h and imaged live with a CellDiscoverer 7 microscope. Imaging and quantifications were done as in (A). Also see Movie EV5.

Source data are available online for this figure.

Figure 6. VHH_mASC ameliorates MSU gouty inflammation

1. Schematic representation of the experimental setting used for the MSU‐gout model. Mice were injected i.a with 100 µg of monosodium urate (MSU) crystals into the knee. After 3 h, mice were treated intraperitonially (i.p.) with VHH_mASC, VHH NP‐1 as unrelated nanobody (both 5 mg kg⁻¹), or vehicle (PBS).

2. Mechanical allodynia threshold and edema were evaluated at 3 and 6 h post‐MSU challenge. Error bars represent SEM from biological replicates: t0, n = 3 for all groups; t3 and t6: PBS + VHH NP‐1 and PBS + VHH_mASC, n = 3 (Mechanical threshold) or n = 4 (Edema); MSU + Vehicle, n = 4; MSU + VHH NP‐1, n = 7; MSU + VHHmASC, n = 7.

3. Flow cytometric assessment of infiltrating total leukocytes (CD45⁺) and granulocytes (CD45⁺ Ly6G⁺ Ly6C⁻) recovered in the synovial fluid of the knee joints of mice treated as in (A). Data in (C) is displayed as floating bars with the max/min values and mean (thicker band). Biological replicates are: PBS + VHH NP‐1, n = 4; PBS + VHH_mASC, n = 4; MSU + Vehicle, n = 4; MSU + VHH NP‐1, n = 7; MSU + VHH_mASC, n = 7.

4. ELISA of IL‐1β, TNFα and IL‐6 in tissue homogenates of knee joints of mice treated as in (A). Data in (C) is displayed as floating bars with the max/min values and mean (thicker band). Biological replicates are PBS + VHH NP‐1, n = 4; PBS + VHH_mASC, n = 4; MSU + Vehicle, n = 4; MSU + VHH NP‐1, n = 7; MSU + VHH_mASC, n = 7.

Data information: ^ns P > 0.05; *P < 0.05; **P < 0.005, One‐way ANOVA, multiple comparison (Tukey test). Data with non‐normal distribution were tested with Krustal‐Wallis test and multiple comparison using Dunn’s test. Outlier in (D, IL‐1β) was determined by the ROUT method and are represented with Δ.

Figure 7. VHH_mASC ameliorates antigen‐induced arthritis

1. A

   Schematic representation of the experimental setting used for the mBSA‐induced arthritis model. Mice were injected sub‐cutaneously (s.c.) with mBSA (500 µg/animal) in an emulsion containing 1 mg ml⁻¹ Freund’s adjuvant on day 0 and day 7. Control mice received injections lacking mBSA. Mice were then immunized with an intra‐articular injection of mBSA (100 µg, right knee) on days 21 and 26. From day 21 until day 26, mice were treated daily with an intra‐peritoneal (i.p.) injection of VHH_mASC (5 mg kg⁻¹), IL‐1RA (50 µg kg⁻¹), or vehicle (PBS).

2. B, C

   Mechanical allodynia threshold (B) and edema (C) were evaluated on day 27. Data is displayed as floating bars with the max/min values and mean (thicker band). Biological replicates are: PBS + Vehicle, n = 4; PBS + VHH_mASC, n = 3; mBSA + Vehicle, n = 6; mBSA + VHH_mASC, n = 6; mBSA + IL‐1RA, n = 5.

3. D

   Flow cytometric assessment of infiltrating total leukocytes (CD45+), granulocytes (CD45⁺ Ly6G⁺ Ly6C⁻), and inflammatory monocytes (CD45⁺ Ly6G⁻ Ly6C⁺) recovered in the synoival fluid of the knee joints of mice treated as in (A). Data is displayed as floating bars with the max/min values and mean (thicker band). Biological replicates are: PBS + Vehicle, n = 4; PBS + VHH_mASC, n = 3; mBSA + Vehicle, n = 6; mBSA + VHH_mASC, n = 6; mBSA + IL‐1RA, n = 5.

4. E

   ELISA of IL‐1β, TNFα and IL‐6 in tissue homogenates of knee joints of mice treated as in (A). Data is displayed as floating bars with the max/min values and mean (thicker band). Biological replicates are: PBS + Vehicle, n = 4; PBS + VHH_mASC, n = 3; mBSA + Vehicle, n = 6; mBSA + VHH_mASC, n = 6; mBSA + IL‐1RA, n = 5.

Data information: ^ns P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, One‐way ANOVA, multiple comparison (Tukey test). Data with non‐normal distribution were tested with Krustal–Wallis test and multiple comparison using Dunn’s test. Outliers were determined by the ROUT method and are represented with Δ.