Unless noted, all reagents were from Thermo Fisher Scientific (Waltham, MA, USA). FCA, nigericin, and Ponceau S were from Sigma-Aldrich (St. Louis, MO, USA). PV was from TCI America (Portland, OR, USA). Ac-YVAD-cmk was from Enzo Life Sciences (Farmingdale, NY, USA). Fam-YVAD-fmk (FLICA) was from ImmunoChemistry Technologies (Bloomington, MN, USA). Ultrapure LPS was from Invivogen (San Diego, CA, USA). One endotoxin unit per milliliter ultrapure LPS is approximately equal to 1 ng/mL from the same manufacturer. GelRed was from Phenix Research Products (Candler, NC, USA). SLO was purified, and hemolytic activity was assessed as previously described (49). The specific activity of SLO was 1.07 × 10⁶ HU/mg. Murine IL-1β cloned into pFB-Neo retroviral vector (Agilent, Santa Clara, CA, USA) using NotI and EcoRI was obtained from Dr. Chenqun Sun (University of Pittsburgh). Murine Casp1 was cloned into peCFP-N1 (Clontech, Mountain View, CA, USA) using XhoI and NotI, which removed the fluorescent protein. Human Dnase1L3 was cloned into pIRES-DsRed (Clontech) using EcoRI and BamHI sites. The R206C mutation was made in Dnase1L3 via Quikchange mutagenesis (Agilent, Santa Clara, CA, USA). PCR primers were from Integrated DNA Technologies (Coralville, IA, USA) with sequences available upon request.

The anti-HMGB1 mouse monoclonal antibody (mAb, Catalog: ab79832) was from Abcam (Cambridge, MA, USA). The anti-β-actin mAb AC-15 (Catalog: 612657) was from BD Biosciences (Franklin Lakes, NJ, USA). The anti-IL-1β mAb 3ZD was from the Frederick National Laboratory for Cancer Research (Frederick, MD, USA) (50). The anti-Casp1 mAb Casper-1 (Catalog: AG-20B-0042-C100) was from AdipoGen (San Diego, CA, USA). Anti-ASC mAb TMS-1 (Catalog: 653902), capture and detection anti-TNFα antibodies (Catalog 79092 and 78335, respectively), capture and detection anti-human IL-1β antibodies (Catalog: 508302 and 508202, respectively), streptavidin conjugated to HRP (Catalog 405210), and TMB substrate (Catalog 421101) were from BioLegend (San Diego, CA, USA). Capture and detection anti-murine IL-1β antibodies (Catalog: 14-7012 and 13-7112, respectively) were from eBioscience (San Diego, CA, USA). Anti-Lamin A/C (MANLAC-4A7-s) was deposited by G.E. Morris (DSHB Hybridoma Product MANLAC1(4A7)) to the Developmental Studies Hybridoma Bank, which was created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA, USA. Fluorescently conjugated secondary antibodies (Catalog: M31501) were from Thermo Fisher Scientific. HRP-conjugated secondary antibodies (anti-mouse 715-035-151; anti-rabbit 711-035-152) were from Jackson Immunoresearch (West Grove, PA, USA).

All mice were housed and maintained at the University of Pittsburgh or Texas Tech University according to respective IACUC standards. Bone marrow from NLRP3^−/− mice was a generous gift from Dr. Tim Billiar, while bone marrow from Casp1^−/− mice was a generous gift from Dr. Richard Flavell, and bone marrow from C57BL/6 mice was a generous gift from Dr. Lisa Borghesi. Additional mice were purchased from The Jackson Laboratory (stock # 000664, 021302, and 016621 for C57BL/6, NLRP3^−/−, and Casp1^−/−, respectively). Mice of both gender aged 6–15 weeks were used to prepare BMDM. The source of the bone marrow did not alter the results. Sample size was determined as the minimum number of mice needed to provide sufficient bone marrow for the experiments. Consequently, no randomization or blinding was needed.

Bone marrow-derived macrophages (BMDM) were isolated and cultured as previously described (19, 33). THP1 cells (ATCC TIB-202) were cultured at 37°C in DMEM supplemented with 10% FCS and 1× l-glutamine (D10) and 1× penicillin/streptomycin and differentiated for 2 days using 5 ng/mL phorbol 12-myristate 13-acetate (PMA) prior to assay. HEK cells (ATCC CRL-1573) were cultured in D10 at 37°C. All cell lines were negative for mycoplasma.

THP1 cells were transfected using Lipofectamine RNAiMAX and 10 pM siRNA (Dnase1L3 HSS 102839) pools directed against Dnase1L3 or control siRNAs (Catalog: 1691553) for 2 days in the presence of 5 ng/mL PMA. Pools of multiple Dnase1L3 siRNAs were used instead of individual siRNAs to reduce the potential for off-target effects (51). RNAi efficiency was evaluated by comparing mRNA expression and phenotype between specific Dnase1L3 siRNAs and non-specific, control siRNAs, in accordance with previously accepted RNAi guidelines (52). HEK cells were plated at 2.5 × 10⁵ cells/well 1 day prior to transfection with 500 ng of indicated plasmids using Lipofectamine2000 and incubated at 37°C either overnight (IL-1β/Casp1) in the presence of nothing, 100 µM FCA, or 20 µM PV for 2 days (Dnase1L3) at 37°C at which point in time supernatants were collected for ELISA or cell lysates generated.

Total RNA was isolated using TriReagent, according to the manufacturer’s instructions. RNA was used for cDNA synthesis with SuperScript^® III reagents. Quantitative PCR on duplicate samples was performed using the SYBR^® Select Master Mix and normalized to the housekeeping gene GAPDH. Primers used were 5′-GATCATCAGCAATGCCTCCT-3′ and 5′-TGTGGTCATGAGTCCTTCCA-3′ for human GAPDH; 5′-AGCCCTTTGTGGTCTGGTTC-3′ and 5′-TCCTTAACGGATGTCTCTGGG-3′ for human Dnase1L3; 5′-GAAATCGTGCGTGACATCAAAG-3′. Relative expression of Dnase1L3 was calculated using the ΔΔCt standardization method.

Supernatants were trichloroacetic acid (TCA) precipitated (50), and cells were lysed in SDS sample buffer, denatured, boiled for 5 min, resolved by SDS-PAGE, and proteins transferred onto a 0.45-µm nitrocellulose membrane. Immunoblotting with primary antibodies Casper-1 anti-Casp1 (1:3,000), 3ZD anti-IL-1β (1:1,000), EPR3057 anti-HMGB1 (1:3,000), MANLAC1 anti-Lamin A/C (1:1,000), TMS-1 anti-ASC (1:1,000), and AC-15 anti-β-actin (1:3,000) was followed by secondary anti-rabbit or anti-mouse antibodies conjugated to HRP (1:10,000). Antibody staining was visualized on a FluorChemE (Protein Simple, San Jose, CA, USA) using enhanced chemiluminescence reagent [0.01% H₂O₂ (Walmart, Bentonville, AR, USA), 0.2 mM p-Coumaric acid (Sigma-Aldrich), 1.25 mM Luminol (Sigma-Aldrich), and 0.1 M Tris, pH 8.4]. Immunoblots were analyzed using Photoshop Creative Suite 3 (Adobe, San Jose, CA, USA). The images were inverted, and integrated intensity of the bands was measured. The percentage of IL-1β or HMGB1 in the supernatant was ratioed to pro-IL-1β or HMGB1 in the lysate and then expressed as a percentage of the control. Pro-IL-1β and lysate HMGB1 levels were used because the actin loading control was saturated in many experiments. In those experiments where actin was not saturating, normalizing to actin instead of pro-IL-1β or HMGB1 did not alter the results.

BMDM (10⁵ for ELISA or immunofluorescence and 10⁶ for immunoblot) were primed with 100 EU/mL ultrapure LPS for 4 h, washed in PBS, and stimulated with 20 µM nigericin or 0.5 kHU/mL SLO for 0–30 min in RPMI at 37°C. This SLO dose is sublytic in macrophages (19). BMDM were incubated with inhibitors for 30 min prior to and during inflammasome stimulation at the following concentrations: 100 µM FCA, 20 µM PV, 50 mM KCl, and 100 µM YVAD. Inhibitor stock solutions were 10 mM FCA in ethanol, 10 mM PV in water, 4 M KCl in water, and 50 mM YVAD in DMSO. Equivalent volumes of ethanol or DMSO had no effect in any experiments performed. For Salmonella enterica typhimurium infection, BMDM were LPS primed for 4 h prior to infection at an MOI of 50 for 1 h. BMDM were incubated for 1 h with FLICA in some experiments along with either SLO or nigericin. The additional time was necessary since FLICA is based on YVAD, which binds to the active site of Casp1 and slows inflammasome activation at the doses of FLICA used.

Cells were fixed for 15 min in 2% paraformaldehyde; permeabilized/blocked for 15 min in 0.1% Triton X-100, 10% goat serum, and 0.05% saponin; stained with EPR3057 anti-HMGB1 mAb or TMS-1 anti-ASC mAb for 1 h, followed by goat anti-rabbit-Alexa 488 or goat anti-rabbit-Cy5 and goat anti-mouse Cy3 for 1 h, and finally DAPI stained. Cells were imaged by epifluorescence microscopy using either an Olympus Provis equipped with 20× (0.85 NA) or 60× (1.42 NA) objective lenses running Magnafire 2.1 (Olympus, Center Valley, PA, USA) or an Olympus BX41 equipped with 60× (1.40 NA) objective running QCapture Pro 7 (QImaging, Surrey, BC, Canada). Nuclear HMGB1 egress was calculated using Metamorph (Molecular Devices, Sunnyvale, CA, USA) to determine the average nuclear intensity of HMGB1. HMGB1 low cells were determined to be those cells containing less nuclear fluorescence intensity than 80–90% of untreated cells in the same experiment. The background levels of untreated cells were then subtracted from each condition. ASC speck formation in THP1 cells was quantitated using ImageJ (53).

Cytokine levels in cell culture supernatants were determined by ELISA. TNFα ELISA was performed according to the manufacturers’ instructions. IL-1β ELISA was performed as previously described (19, 33) although murine IL-1β capture and detection antibody concentrations were 2 and 3 µg/mL, respectively, and human IL-1β capture and detection antibody concentrations were 3 and 2 µg/mL, respectively.

HEK cells were grown for 24 h at 2.5 × 10⁵ cells per well. The cells were transfected with Dnase1L3 or Dnase1L3 R206C or left untreated and incubated at 37°C, 5% CO₂ for 24 h. The cells were harvested with trypsin, centrifuged at room temperature for 5 min at 1,000 × g, washed with PBS, and centrifuged again. The cell pellets were resuspended in Dnase assay buffer (20 mM Tris, pH 7.4, 5 mM MgCl₂, 2 mM CaCl₂) with 1% Triton X-100 and 0.5% phenylmethylsulfonyl fluoride and then kept on ice for 10 min. The cells were then centrifuged at 4°C for 15 min at 17,000 × g. The supernatants were saved for Dnase activity assays. Dnase1L3 activity was determined by incubating 400 ng plasmid DNA with cell lysates in the presence or absence of 50 µM FCA, PV, or aurintricarboxylic acid (ATA) in 20 mM Tris, pH 7.4, 5 mM MgCl₂, 2 mM CaCl₂ for 30 min at 37°C. The extent of degradation was quantitated using Photoshop Creative Suite 3 from Gel Red-stained agarose gels. Transfection efficiency was determined by flow cytometry, and equal transfection was observed between Dnase1L3 wild type (WT) and R206C. Dnase1L3 activity was determined by titrating lysates until 50–70% degradation was observed. This dilution was then used to determine inhibitor activity.

BMDM were primed for 4 h at 37°C with 100 ng/mL LPS, harvested, incubated with various concentrations of SLO in RPMI with 20 µg/mL propidium iodide (PI) for 5 or 30 min at 37°C and analyzed by FACS on an LSR II (BD Biosciences). Debris, defined as any event with a subcellular forward and side scatter profile, was gated out, and the percentage of cells in the PI-high gate was determined. Specific lysis was determined by subtracting the background from each point and dividing by 100 less the background. LDH assays were performed according to manufacturer’s instructions.

BMDM (10⁶) were treated with SLO at various concentrations for 30 min at 37°C or UV irradiated for 15 s followed by 4 h culture at 37°C. THP1 (5 × 10⁵) cells were either treated with SLO at various concentrations for the indicated time points or treated with 15 µM etoposide (Etop) for 6 h at 37°C. Following treatment, cells were stained with 0.9 µg/mL Annexin V-FITC and 20 µg/mL PI in Annexin V Binding Buffer (1.4 mM NaCl, 25 mM CaCl₂, 100 mM HEPES, pH 7.4) for 15 min on ice and analyzed by FACS on an Accuri C6 (BD Biosciences). Debris, defined as any event with a subcellular forward and side scatter profile, was gated out, and the percentage of PI⁺, Annexin V⁺, or double-positive cells was determined.

Prism 5.0 (GraphPad, La Jolla, CA, USA) software was used for statistical analysis. Data are represented as mean ± SEM as indicated. Statistical significance was determined by one-way or two-way ANOVA with Bonferroni posttesting; p < 0.05 was considered statistically significant.

We investigated whether Dnase1L3 was needed for IL-1β secretion in B6 BMDM using Dnase1L3 inhibitors. We tested whether the Dnase1L3 inhibitors, FCA and PV, blocked inflammasome-dependent IL-1β production. To trigger inflammasome activation, we used either the prototypical NLRP3 agonist, nigericin, or the more biologically relevant, tunable NLRP3 agonist, SLO. We treated LPS-primed BMDM with either sublytic SLO or nigericin and measured IL-1β secretion in the absence or presence of FCA, PV, or the Casp1 inhibitor YVAD. Similar to YVAD-mediated inhibition, both FCA and PV potently blocked IL-1β secretion (Figure 1A). Since FCA blocked IL-1β release, it is possible that FCA generally blocked cytokine release. To test this hypothesis, we next measured the levels of TNFα secreted by the LPS-primed BMDM following inhibitor treatment during inflammasome activation. We found that NLRP3 inflammasome activation did not reduce TNFα secretion (Figure 1B). Furthermore, treatment with either YVAD or FCA did not reduce TNFα levels (Figure 1B). These findings demonstrate that inhibition of Dnase1L3 does not block general cytokine release but blocks IL-1β release in primary B6 BMDM stimulated with NLRP3 inflammasome activators.

The IL-1β blockade we observed by FCA or PV treatment could be due to direct inhibition of Casp1 via inhibitor off-target effects. To test this hypothesis, we transfected HEK cells with IL-1β and Casp1. When Casp1 is overexpressed in HEK cells, it autoactivates without NLRs or ASC and processes any pro-IL-1β present in the cells (54, 55). When we treated transfected HEK cells with either FCA or PV, we found no change in IL-1β secretion (Figure 1C). These results indicate that Casp1 is not a direct target of these Dnase1L3 inhibitors.

We next confirmed that FCA and PV inhibit Dnase1L3 activity by expressing either WT or R206C Dnase1L3 in HEK cells. The R206C mutation eliminates Dnase1L3 activity (8). To control for transfection efficiency between experiments, we titrated the Dnase1L3 activity to degrade ~60% of a fixed amount of plasmid DNA. Transfected Dnase1L3 was the only source of Dnase1L3 activity in HEK lysates, and this activity was eliminated by the R206C mutation (Figures 1D,E). When lysates were incubated with either FCA or PV, we observed a reduction in Dnase1L3 activity similar to that provided by the R206C mutation (Figures 1D,E). The inhibition was also similar to that observed with the general nuclease inhibitor ATA (Figures 1D,E). These findings suggest that FCA and PV block Dnase1L3 activity.

Another potential explanation for our finding that Dnase1L3 inhibition blocked IL-1β release is that the inhibitor targets SLO-dependent cell death cells independently of inflammasome activation prior to IL-1β release (19). Since SLO challenge is associated with pyroptosis, necrosis, and apoptosis (18, 19, 56), we determined the dose of SLO needed to kill human THP1 cells and primary B6 BMDM and the mechanism of cell death. We measured necrosis using PI uptake and simultaneously we measured apoptosis using Annexin V. Following 30-min SLO treatment, we did not detect any apoptosis in BMDM or THP1 cells (Figures 2A,B). In contrast, UV irradiation or etoposide treatment for longer periods of time promoted apoptosis in BMDM and THP1 cells (Figures 2A,B). Consistent with previous results (19), we found that unprimed BMDM were more resistant to lysis by SLO than other cell types, including THP1 cells (Figures 2A,C). For BMDM, 0.5 kHU/mL was determined to be a sublytic dose, while 4–5 kHU/mL provided a lytic dose (Figure 2A). In contrast, the sublytic dose for THP1 cells was found to be 0.125 kHU/mL and lytic dose to be 0.5 kHU/mL (Figure 2C). Thus, SLO promoted neither apoptosis nor necrosis at sublytic concentrations.

We tested whether sublytic SLO challenge promotes inflammasome activation by comparing Casp1 activation and HMGB1 secretion in WT, NLRP3^−/−, and Casp1^−/− BMDM treated with sublytic or lytic doses of SLO. We found that in WT BMDM, sublytic SLO doses activated Casp1 and depleted HMGB1 from cell lysates, resulting in extracellular HMGB1 release (Figure 2D). In contrast, lytic SLO doses released HMGB1 without Casp1 activation (Figure 2D). In NLRP3^−/− and Casp1^−/− BMDM, sublytic SLO promoted minor HMGB1 egress, without appreciably depleting cytoplasmic stores (Figure 2D). These results indicate that robust HMGB1 egress under these conditions requires inflammasome activation. In contrast, lytic SLO doses promoted HMGB1 release independently of NLRP3 or Casp1 activation (Figure 2D). Thus, sublytic SLO can promote HMGB1 release and Casp1 activation in a NLRP3 inflammasome-dependent manner. Furthermore, SLO dose does not account for the FCA- or PV-mediated IL-1β blockade.

We next tested whether FCA blocks HMGB1 release. Since nuclear egress seemed to be a more sensitive readout of inflammasome-dependent activation than presence of HMGB1 in the supernatant (Figure 2D), we measured nuclear HMGB1 levels by immunofluorescence. We treated LPS-primed BMDM from WT and NLRP3^−/− mice with sublytic SLO or nigericin for 10–30 min and measured HMGB1 release (Figures 3A,B). We found that 4 h of LPS priming was insufficient to promote HMGB1 export, as previously described (35), but that subsequent sublytic SLO or nigericin treatment rapidly promoted the release of nuclear HMGB1 (Figures 3A,B). We found that HMGB1 egress was reduced in NLRP3^−/− BMDM, indicating HMGB1 release under these conditions was NLRP3 dependent (Figure 3B). We next used this system to determine whether Dnase1L3 contributes to inflammasome-dependent HMGB1 release during toxin challenge. We treated LPS-primed BMDM with 100 µM FCA followed by sublytic SLO challenge and measured HMGB1 nuclear egress (Figure 3C). Compared to the control, the level of HMGB1 release from FCA-treated cells was significantly reduced (Figure 3C). Thus, robust HMGB1 release following NLRP3 inflammasome activation requires Dnase1L3 nuclease activity.

Since it appeared that FCA blockade reduced both IL-1β secretion and HMGB1 release, we next tested whether Dnase1L3 is needed for Casp1 cleavage. We treated WT, Casp1^−/−, or NLRP3^−/− LPS-primed BMDM with sublytic SLO or nigericin with or without YVAD or FCA and examined processing and release of IL-1β and Casp1. Mature IL-1β and Casp1 p20 were detected in the TCA-precipitated supernatant of WT BMDM (Figure 4A). In contrast, both FCA and YVAD reduced mature IL-1β or active Casp1 levels to that observed in Casp1^−/− and NLRP3^−/− BMDM (Figure 4A). There was slight, variable toxicity to the cells from FCA, as measured by pro-IL-1β release in FCA-treated knockout BMDM and a slight (but not statistically significant) decrease in lysate levels of actin and other proteins in the cell lysate in WT, NLRP3^−/−, and Casp1^−/− BMDM (Figure 4B). Although IL-1β levels were decreased tremendously following inhibitor treatment or in the absence of NLRP3 or Casp1, HMGB1 egress was reduced ~50% following treatment with YVAD or FCA (Figures 4C,D). This impairment was somewhat variable, though generally ~50%. The incomplete inhibition of HMGB1 release by these assays could be due to cell lysis. Overall, we find that Dnase1L3 activity is required for IL-1β and Casp1 processing.

Since HMGB1 is released by cell lysis (Figure 2D), it is possible that FCA does not significantly impair pyroptosis. We next tested whether Dnase1L3 is required for pyroptosis. Pyroptosis typically occurs after 30 min of nigericin or SLO treatment, kinetically similar to IL-1β release (19) and is usually measured via membrane integrity assays in the presence and absence of inflammasome inhibitors or in Casp1/11 knockout cells. For consistency with previous results (19), we chose to use SLO to measure pyroptosis. Since lysis due to SLO is also measured via membrane integrity, we took advantage of the kinetic differences between direct SLO lysis and pyroptosis to distinguish between pyroptosis and direct SLO-mediated lysis. We treated LPS-primed BMDM with SLO for 5 min to assess direct SLO cytotoxicity or 30 min to allow time for pyroptosis. We measured specific cell lysis by PI uptake. FCA increased baseline sensitivity from 21% ± 3.79 (mean ± SEM) in control BMDM to 35.3% ± 1.65 (p = 0.055) at 30 min, indicating slight toxicity to cells from FCA. When challenged with SLO, WT, NLRP3^−/−, and Casp1^−/− BMDM showed equivalent sensitivity at 5 min, indicating that ablation of inflammasome components does not alter cell sensitivity to direct pore formation (Figure 5A). However, by 30 min, the extreme sensitivity of WT BMDM to SLO was revealed (Figure 5A). This extreme sensitivity was due to pyroptosis, since NLRP3^−/− and Casp1^−/− BMDM did not show similarly increased sensitivity (Figure 5A). Likewise, inhibition of NLRP3 inflammasome activation by KCl blocked pyroptosis (Figure 5B). Using this system, we tested the requirement for Dnase1L3 in pyroptosis. Unlike KCl, FCA only mildly impaired pyroptosis, leaving BMDM 2-fold less sensitive to SLO, compared to the 20-fold reduction observed with KCl (Figure 5B). Thus, Dnase1L3 inhibition can uncouple NLRP3-induced cytokine secretion from pyroptosis.

Since Dnase1L3 activity was necessary for NLRP3-dependent Casp1 activation and IL-1β and HMGB1 release, we tested whether the requirement for Dnase1L3 is specific to NLRP3 or also required for other inflammasomes. We tested the NLRC4 inflammasome, since it can engage Casp1 directly (57). To test the role of Dnase1L3 during NLRC4 activation, we measured IL-1β release by ELISA from LPS-primed BMDM infected with Salmonella enterica typhimurium for 1 h. At this time point, S. typhimurium specifically triggers NLRC4-dependent, but NLRP3-independent inflammasome activation (30–32, 58). Under NLRC4-activating conditions, we found that FCA and PV reduced IL-1β release compared to the control (Figure 6A). Similarly, FCA and PV blocked mature IL-1β production and Casp1 cleavage following S. typhimurium infection (Figure 6B). Following quantitation of the western blots, we found that while IL-1β was potently blocked, HMGB1 egress was variably decreased following FCA or PV treatment (Figure 6C). This is consistent with the relatively mild ability of Dnase1L3 inhibitors to impair pyroptosis (Figure 5). Overall, we conclude that Dnase1L3 activity is also important for Salmonella typhimurium-induced Casp1 and IL-1β processing.

Since pharmacological inhibitors can have off-target effects, and because FCA and PV are poorly characterized, we knocked down Dnase1L3 using RNAi in PMA-differentiated THP1 cells. PMA-differentiated THP1 cells are a human model system for inflammasome activation (59), so we used the model NLRP3 agonist nigericin to measure inflammasome activation. The mRNA knockdown efficiency of Dnase1L3 expression was typically ~90% (Figure 7A). We found that similar to the inhibitors, ablation of Dnase1L3 reduced IL-1β secretion by ELISA (Figure 7B). We tested whether pyroptosis was impaired by LDH release and found that this assay could not detect any significant change in pyroptosis between untransfected and Dnase1L3 siRNA-transfected cells (Figure 7C). Dnase1L3 RNAi reduced mature IL-1β processing compared to control cells (Figure 7D). Since there was some variability in cell numbers, we quantitated our western blots and ratioed IL-1β secretion and HMGB1 release to levels in the cytosol. We found that HMGB1 egress was ~75% blocked and IL-1β ~90% blocked compared to the control group (Figure 7E). Mock siRNA did not reduce IL-1β or HMGB1 release to similar levels, suggesting that the inhibition was specific to Dnase1L3 (Figure 7E). We conclude that Dnase1L3 is necessary for inflammasome-mediated cytokine release based on both genetic and inhibitor experiments.

To examine the mechanism through which Dnase1L3 potently regulates cytokine release, but only mildly affects pyroptosis, we examined ASC release and speck formation. Formation of ASC specks is required for efficient cytokine IL-1β secretion by both NLRP3 and NLRC4 inflammasomes (30, 32). In contrast, the absence of ASC speck formation reduces pyroptosis only twofold (32, 57). This is nearly identical to the phenotype we observed following Dnase1L3 blockade. We measured ASC speck formation in PMA-differentiated THP1 cells following Dnase1L3 RNAi and inflammasome activation. ASC speck formation induced by nigericin was abrogated following Dnase1L3 RNAi, but unperturbed in mock transfected and untransfected cells (Figures 8A,B). Under these conditions, we detected an average of one ASC speck for every two cells (Figure 8B). Following Dnase1L3 RNAi, this dropped to one ASC speck per 26 cells (Figure 8B). Finally, we tested whether ASC levels were perturbed by Dnase1L3 RNAi. We found that ASC levels relative to actin were not significantly altered by Dnase1L3 siRNA (Figure 8C). Overall, these data suggest that Dnase1L3 is necessary for ASC speck formation.

We next tested whether Dnase1L3 inhibition blocked ASC speck formation and Casp1 recruitment in primary BMDM using the more biologically relevant NLRP3 agonist, SLO. In LPS-primed cells, ASC localized to the nucleus (Figure 8D). Following inflammasome activation, we observed ASC recruitment to one to three specks in a subset of BMDM (Figure 8D). Not all cells contained specks, suggesting heterogeneity in inflammasome activation by SLO and consistent with our findings in THP1 cells with nigericin (Figure 8B). We tested whether these specks were sites of active inflammasome assembly by visualizing active Casp1 in these cells. Active Casp1 in SLO-treated BMDM cells was visualized using FLICA, a fluorogenic Casp1 substrate. Active Casp1 was present in the cytosol and colocalized with ASC in specks (Figure 8D). Only cells that formed specks were depleted of nuclear HMGB1, indicating secretion of HMGB1 (Figure 8E). We did not detect HMGB1 in inflammasome specks (Figure 8E). In contrast, BMDM pretreated with FCA showed only diffuse cytosolic Casp1 upon SLO exposure and no inflammasome speck formation (Figure 8D). HMGB1 generally remained in the nucleus of FCA-treated cells (Figure 8E). This suggested that FCA blocked recruitment of ASC and Casp1 to inflammasome specks. Thus, Dnase1L3 blockade uncouples IL-1β production from pyroptosis by preventing ASC nuclear egress and speck formation.