Active caspase-1 and ASC release in microparticles from stimulated THP-1.

<p>THP1 cells were stimulated with LPS (1μg/ml) for 2h in the presence or absence of YVAD-cmk. Microparticles were isolated from LPS stimulated THP-1 supernatant by stepwise ultracentrifugation (A). The cell fraction was separated by centrifugation at 2000 g for 5 min. Supernatants were then further fractionated into a 16,000g fraction followed by microparticle (MP) fraction and non- microparticle (nonMP) fraction by a final ultracentrifugation at 100,000 g for 1h. Microparticles (MP) and non-microparticle fractions (nMP) were analyzed for presence of the p20 form of caspase-1 (B) and the inflammasome adaptor protein, ASC (C) by immunoblot. Substrate cleavage capacity of caspase-1 from each of the supernatant fraction was also measured using WEHD-enzymatic assay (D). Analysis for n = 2 experiments.</p

Encapsulation is necessary for exogenous caspase-1 mediated apoptosis.

<p>THP1 cells were first labeled with phospholipid dye, DiLC16 for 30 min, washed, stimulated with LPS (1μg/ml) for 2h and microparticles were isolated. A) Labeled microparticles were observed under fluorescence microscope; left panel (top and bottom). Uptake of labeled microparticles by DAPI stained HPMVEC was observed by fluorescence microscopy (right panel: control MP (top) and LPS MP (bottom)). B) Microparticles were then either kept intact or disrupted by vortexing or by sonication, or subjected to heat inactivation. Microparticles were then applied to HPMVECs and endothelial cell viability was analyzed by MTS assay (n = 3). * intact LPS MP vs control MP or intact LPS MP vs ruptured/ heat inactivated LPS MPs. C) Caspase-1 activity of each fraction was measured using WEHD enzymatic assay from two experiments. Mild homogenization of microparticles or sonication did not affect caspase-1 activity in contrast to heat inactivation. D) HPMVEC were pretreated with cytochalasin D (5μg/ml) for 30 min, washed two times to remove trace of reagent and then control or LPS MP was subjected to cells as previously described. Uptake of microparticles and effect on apoptosis of HPMVEC was analyzed by MTS assay (n = 3 experiments) * intact LPS MP vs control MP; # LPS MP with cytochalasin D vs LPS MP.</p

Human pulmonary microvascular endothelial cell apoptosis is mediated by THP-1 stimulated microparticles and inhibited by caspase-1 inhibitor.

<p>THP1 cells were stimulated with LPS (1μg/ml) for 2h and supernatants were separated into microparticle (MP) and non- microparticle (nonMP) fractions by serial centrifugations. HPMVECs were co-cultured with these MP and nonMP fractions and analyzed for cell viability and apoptosis. Cell viability was measured by MTS assay (A and B) and morphology using DAPI staining (C). HPMVECs were then treated with microparticles isolated from unstimulated or LPS induced THP1 in the presence or absence of the caspase-1 inhibitor, YVADcmk (D). Apoptosis was analyzed using Annexin V/PI assay by flow cytometry. Quantitative analysis for n = 3 experiments. * #Comparison of LPS MP to control MP/cells only, $ Comparison of LPS MP to LPS nonMP, @ Comparison of LPS MPs, with and without YVAD.</p

A1AT does not influence IL-18 processing and release through the activation of caspase-1.

<p>THP-1 cells and human monocytes were pre-treated with A1AT (2.5 mg/mL) for 1 h followed by LPS 1 μg/ml for 30 minutes, and then triggered with ATP 5 mM for another 30 minutes to determine the release of IL-18 and caspase-1. Same concentration of BSA was used as control. <b>A.</b> THP-1 cell and <b>B.</b> Human monocytes, culture media were assayed by ELISA for the released IL-18, and by immunoblots for IL-18 and caspase-1. ELISA data represent the means ± SD for three independent experiments in THP-1 cells and human monocytes, and the blots are representative of repeated blots. NS indicates no significant difference between BSA control and A1AT treatment, as analyzed by Student’s t-test.</p

Effect of A1AT on caspase-1 activation in THP-1 cells culture.

<p>THP-1 cells pretreated with A1AT (0.5 to 2.5 mg/ml), then LPS 1 μg/ml stimulated for 30 minutes followed by 5 mM ATP challenge for another 30 minutes. Caspase-1 activation was measured both in lysate and supernatant by using caspase-1 fluorogenic tetrapeptide substrate (WEHD-afc) according to materials and methods. Data represent the mean ± SD for three independent experiments.</p

A1AT is ineffective on endogenous caspase-1 activation in cell-free system.

<p>Caspase-1 activity was measured in cell-free lysates from THP-1 monocytes. Cell-free lysates in a hypotonic buffer were placed at 4 or 30°C for 1 hour with or without A1AT (2.5 mg/mL). Same concentration of BSA was used as control. Caspase-1 activity was then measured by using caspase-1 fluorogenic tetrapeptide substrate (WEHD-afc) according to materials and methods. Data represent the means ± SD for three independent experiments. NS indicates no significant difference between BSA control and A1AT, as analyzed by Student’s t-test.</p

Release of mature caspase-1 is ASC dependent but extracellular caspase-1 activity does not co-precipitate with ASC or predominate in microvesicles.

<p>A) THP1 (2x10⁷ cells/ml) cells stably transduced with shRNA for ASC (siASC) and EGFP (siEGFP control) were left untreated or stimulated with LPS/ATP (L/A) for 1h and caspase-1 activity from the supernatants was measured. B) Lysates and supernatants from <i>(A)</i> were probed for caspase-1, ASC, and β-actin by immunoblot. C) Supernatants of LPS/ATP treated THP1 cells (2x10⁷ cells/ml) were analyzed for WEHD-afc activity after immunodepletion with either monoclonal anti-ASC antibody (α-ASC) or control mouse IgG1A (IgG) (top panel). Immunoblots for caspase-1 and ASC confirm effective ASC depletion (bottom panel), immunoprecipitate beads <b>(B)</b> and residual supernatant <b>(Sup)</b>. Co-immunoprecipitation of caspase-1 and immunoprecipitation of ASC are shown below. Original supernatant (-), immunoprecipitate beads (B) and residual supernatant (Sup). * indicates IgG light chain. D) Supernatants of LPS/ATP treated THP1 cells (2x10⁷ cells/ml) were subjected to microvesicle purification by differential centrifugation. Sup1 (control, 16,000g supernatant), Sup2 (100,000g supernatant), MV (100,000g pellet). Caspase-1 activity was measured in the MV-enriched (Sup1), MV-depleted (Sup2), and MV pellet. Fractions were run on immunoblot and LAMP-1 was used as a marker to confirm microvesicle enrichment [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142203#pone.0142203.ref037" target="_blank">37</a>]. Representative gel and data expressed as mean ± SEM from n = 3 independent experiments (A, B, C and D).</p

Active caspase-1 is found in a high molecular weight complex.

<p>A) Pooled and concentrated supernatant from LPS/ ATP treated THP1 cells (2x10⁷ cells/ml) were fractionated on a HiPrep 16/60 Sephacryl S-200 HR chromatography column. Fractions (2 ml) were collected and analyzed for WEHD-afc activity, detection of caspase-1 by ELISA, and for total protein concentration in the respective fractions. Gamma-globulin (180 kDa), albumin (66 kDa), and myoglobulin (17 kDa) were run on the column as MW markers as indicated. AFU = arbitrary fluorescent units, AU = absorbance units. B) Immunoblots for mature caspase-1 and ASC in the column fractions (15–41) are shown below. Data and representative blots are from n = 2 experiments.</p

LPS/ATP stimulation induces the intracellular formation of ASC specks as a marker of inflammasome activation.

<p>A) YFP-THP1 cells (10⁶ cells/ml) were treated with LPS (1 μg/ml) for 30 min followed by an additional 30 min incubation with ATP (5 mM) to assess the induction of pyroptosis. Bright-field and fluorescent images showing intracellular YFP-ASC specks and loss of membrane integrity are shown. B) Quantification of ASC speck formation in microscopic field under 200X magnification. Representative images and data expressed as mean ± SEM from n = 5 independent experiments.</p

Caspase-1 activity in cell-extract decreases over time.

<p>A) Rapid cleavage of caspase-1 and IL-18 into their mature forms during incubation of cell extract from 3x10⁸ THP1 cells/ml at 37°C. ASC was used as a loading control. B) Cell-extracts were pre-incubated at 37°C for various time points and then loaded simultaneously into the fluorimeter and caspase-1 activity (measured as maximal slope: AFU/min) was measured. Dashed line denotes half-life. C) YVAD-cmk (50 μM) inhibits caspase-1 activity and cleavage. Cell-extracts were incubated at 4 and 37°C in presence or absence of YVAD-cmk for 1h and WEHD-afc cleavage measured (AFU/min). Non-YVAD-cmk inhibitable baseline activity is indicated below the dashed-line. Immunoblots for caspase-1 activation and cleavage of endogenous IL-18 was used to confirm caspase-1 specific activity. <i>***</i>: <i>p<0</i>.<i>0001(ANOVA)</i>. Representative gels from n = 4 and n = 3 independent experiments (A and C, respectively). Data are expressed as mean ± SEM for n = 4 (B) and n = 3 (C) independent experiments.</p