Plasmocin-dependent removal of mycoplasmal contamination of THP-1 cells.

<p>THP-1 cells, chronically infected with mycoplasma, were treated with Plasmocin (25 µg/ml every 3 days) and aliquots were taken every three to four days for detection of mycoplasma by PCR. DNA from mycoplasma negative (−) and positive (+) cells was served as control (Ctr).</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

Extracellular caspase-1 is able to cleave pro-IL-1β.

<p>THP1 cells (2x10⁹ cells/ml) were left untreated or stimulated with LPS/ATP for 1h and then supernatants were incubated with lysates from HEK293 cells expressing proIL-1β-EGFP for 12h at 37°C in the presence or absence of YVAD-cmk (100 μM) or z-FA-fmk (100 μM apoptotic caspase inhibitor, negative control) and immunoblotted for IL-1β. Representative gel from n = 3 independent experiments. Asp²⁷cleavage product of EGFP-proIL-1β detected.</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

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

LPS/ATP treatment induces rapid release of inflammasome components and pyroptosis.

<p>A) THP1 (2x10⁷ cells/ml) were treated with LPS (1 μg/ml) for 30 min followed by an additional 30 min incubation with ATP (5 mM). Lysates and supernatants were probed for mature forms of caspase-1 and IL-18. (NT = Untreated, L = LPS, A = ATP, L/A = LPS/ATP). β-actin was used as internal control. B) Detection of mature IL-18 release into the supernatant. C) Relative cytotoxicity of treatments by LDH release. <i>***</i>: <i>p< 0</i>.<i>0001</i>, <i>(ANOVA)</i>. Representative gel from n = 3 independent experiments, and data from n = 10 and n = 6 experiments, respectively.</p

Activation of caspase-1 is dependent on protein concentration.

<p>A) Total protein yield (top) and caspase-1 cleavage (bottom) resulteing from 10⁸, 2x10⁸, and 3x10⁸ THP1 cells/ml lysed in hypotonic buffer. To measure caspase-1 activation the cell lysate was incubated at 4°C or 37°C for 1h before immunoblotting. B) Cell-extract from 2x10⁸ and 3x10⁸ THP1 cells/ml were diluted and incubated at 37°C for 1h. Immunoblot shows pro-caspase-1 (45 kDa subunit), intermediate caspase-1 (35 kDa subunit), and mature caspase-1 (20 kDa subunit). C) Caspase-1 activity as measured by WEHD-afc cleavage in 10⁸ and 3x10⁸ THP1 cells/ml. <i>**</i>: <i>p<0</i>.<i>0045</i>, <i>***</i>: <i>p< 0</i>.<i>0001 (ANOVA (A)</i>, <i>t-test (C)</i>. Data are expressed as mean ± SEM for n = 8 (A) n = 9 (C) independent experiments and a representative gel from n = 3 (A), n = 1 (B top) and n = 2 (B bottom).</p

Caspase-1 activity detected extracellularly in whole human blood and purified human monocytes.

<p>A) Whole human blood (1 ml) from healthy donors was either left untreated (NT) or stimulated for 30 min with LPS (1 μg/ml) followed by an additional 30 min with ATP (5 mM) (L/A). Plasma was separated from blood cells and incubated with WEHD-afc (50 μM) for 2 days. Activity was calculated by subtracting background (relative absorbance fluorescence unit, RAFU). B) Human monocytes (peripheral blood mononuclear cells (PBMC)) were purified from healthy donors and treated at 2x10⁷ monocytes/ml with LPS (1 μg/ml) and ATP (5 mM) as in <i>(A)</i>. Supernatants were isolated and incubated with WEHD-afc (50 μM) in the presence or absence of YVAD-cmk (50 μM) to determine caspase-1 specific activity. Mean slopes over 2h are plotted from n = 2 independent experiments (A). Mean slopes over 2h are plotted with SEM from n = 5 independent experiments (B). <i>***</i>: <i>p<0</i>.<i>0001(ANOVA)</i>.</p

Inflammasome Priming Is Similar for <i>Francisella</i> Species That Differentially Induce Inflammasome Activation

<div><p>Inflammasome activation is a two-step process where step one, priming, prepares the inflammasome for its subsequent activation, by step two. Classically step one can be induced by LPS priming followed by step two, high dose ATP. Furthermore, when IL-18 processing is used as the inflammasome readout, priming occurs before new protein synthesis. In this context, how intracellular pathogens such as <i>Francisella</i> activate the inflammasome is incompletely understood, particularly regarding the relative importance of priming versus activation steps. To better understand these events we compared <i>Francisella</i> strains that differ in virulence and ability to induce inflammasome activation for their relative effects on step one vs. step two. When using the rapid priming model, i.e., 30 min priming by live or heat killed <i>Francisella</i> strains (step 1), followed by ATP (step 2), we found no difference in IL-18 release, p20 caspase-1 release and ASC oligomerization between <i>Francisella</i> strains (<i>F</i>. <i>novicida</i>, <i>F</i>. <i>holarctica</i> –LVS and <i>F</i>. <i>tularensis</i> Schu S4). This priming is fast, independent of bacteria viability, internalization and phagosome escape, but requires TLR2-mediated ERK phosphorylation. In contrast to their efficient priming capacity, <i>Francisella</i> strains LVS and Schu S4 were impaired in inflammasome triggering compared to <i>F</i>. <i>novicida</i>. Thus, observed differences in inflammasome activation by <i>F</i>. <i>novicida</i>, LVS and Schu S4 depend not on differences in priming but rather on their propensity to trigger the primed inflammasome.</p></div