<i>Staphylococcus aureus</i> Leukocidin A/B (LukAB) Kills Human Monocytes via Host NLRP3 and ASC when Extracellular, but Not Intracellular

<div><p><i>Staphylococcus aureus</i> infections are a growing health burden worldwide, and paramount to this bacterium’s pathogenesis is the production of virulence factors, including pore-forming leukotoxins. Leukocidin A/B (LukAB) is a recently discovered toxin that kills primary human phagocytes, though the underlying mechanism of cell death is not understood. We demonstrate here that LukAB is a major contributor to the death of human monocytes. Using a variety of <i>in vitro</i> and <i>ex vivo</i> intoxication and infection models, we found that LukAB activates Caspase 1, promotes IL-1β secretion and induces necrosis in human monocytes. Using THP1 cells as a model for human monocytes, we found that the inflammasome components NLRP3 and ASC are required for LukAB-mediated IL-1β secretion and necrotic cell death. <i>S</i>. <i>aureus</i> was shown to kill human monocytes in a LukAB dependent manner under both extracellular and intracellular <i>ex vivo</i> infection models. Although LukAB-mediated killing of THP1 monocytes from extracellular <i>S</i>. <i>aureus</i> requires ASC, NLRP3 and the LukAB-receptor CD11b, LukAB-mediated killing from phagocytosed <i>S</i>. <i>aureus</i> is independent of ASC or NLRP3, but dependent on CD11b. Altogether, this study provides insight into the nature of LukAB-mediated killing of human monocytes. The discovery that <i>S</i>. <i>aureus</i> LukAB provokes differential host responses in a manner dependent on the cellular contact site is critical for the development of anti-infective/anti-inflammatory therapies that target the NLRP3 inflammasome.</p></div

LukAB induces necrotic cell death and secretion of pro-inflammatory cytokines IL-1β and IL-18.

<p>(A) THP1 cells were intoxicated with culture filtrate from <i>S</i>. <i>aureus</i> Newman (10% v/v), isogenic <i>lukAB</i> mutant or culture media for 1 hour. Cells were collected, prepared and imaged by transmission electron microscopy (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004970#sec011" target="_blank">methods</a>). (B) THP1 cells were intoxicated with 1% (v/v) culture filtrate from <i>S</i>. <i>aureus</i> Newman or the indicated isogenic mutants and after 4 hours supernatants were collected and analyzed by immunoblot for HMGB1 release. (C) THP1 cells were intoxicated with purified LukAB at the indicated concentrations for 1 hour and supernatants were analyzed by immunoblot for HMGB1 release. (D) THP1 cells were intoxicated with culture filtrates (1% v/v) from the indicated strains and supernatants were collected and analyzed for secretion of the indicated cytokines. THP1 cells (E) and primary CD14+ human monocytes (F) were primed for production of pro-IL-1β with 500 ng/mL LTA for 3 hours followed by intoxication with LukAB (THP1 with 50 ng/mL and CD14+ monocytes with 30 ng/mL) for 1 hour then supernatants were analyzed for secretion of the indicated cytokines. Error bars represent the mean ± standard error of the mean for at least two independent experiments, each performed in triplicate. Primary cell experiments include three independent donors. Asterisks indicate significance at a <i>p</i>-value of < = 0.05 by Tukey’s multiple comparisons post-test for 1-way or 2-way ANOVA, as appropriate.</p

LukAB produced by extracellular or phagocytized <i>S</i>. <i>aureus</i> kills human monocytes.

<p>(A) THP1 cells were infected with <i>S</i>. <i>aureus</i> Newman strains at an MOI of 10 for 120 min under non-phagocytosing conditions (extracellular infection; see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004970#sec011" target="_blank">methods</a>), stained with the fixable viability dye eFluor 450, a membrane damage and cell death marker, and analyzed by flow cytometry. Representative histogram is shown. (B-D) THP1 shRNA cells were infected with <i>S</i>. <i>aureus</i> Newman, MOI 10 for 120 min, followed by flow cytometry analysis for maximal eFluor 450 staining (B) and FLICA-1 activation (C). (D) Culture supernatants were collected from extracellular infections and analyzed for IL-1β secretion. (E and F) THP1 cells were infected with GFP-expressing <i>S</i>. <i>aureus</i> Newman strains at an MOI of 10 for 45 min under phagocytosing conditions (intracellular infection; see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004970#sec011" target="_blank">methods</a>). (E) THP1 cells were stained with the fixable viability dye eFluor 450. After infection, THP1 cells were analyzed by flow cytometry and GFP-positive THP1 cells were selected, indicative of <i>S</i>. <i>aureus</i> phagocytosis. Maximal eFluor 450 incorporation was gated among GFP-positive cells, indicative of THP1 death by intracellular <i>S</i>. <i>aureus</i>. Very few cells appear in the first plot corresponding to the background autofluorescent uninfected cells. Representative plots are shown. (F) Representative histogram of GFP fluorescence in <i>S</i>. <i>aureus</i> infected and uninfected THP1 cells is shown. (G-H) Purified primary CD14+ human monocytes were infected with <i>S</i>. <i>aureus</i> Newman and USA300-BK18807 strains, along with respective isogenic <i>lukAB</i> mutants, at an MOI of 5 for 45 min then stained with eFluor 450 (G) or FLICA-1 (H), and analyzed by flow cytometry. Graphs reflect the fraction of cells that were GFP positive. (I-L) THP1 shRNA cells were infected under phagocytosing conditions with <i>S</i>. <i>aureus</i> Newman, MOI 10 for 45 min, and analyzed by flow cytometry for (I) GFP fluorescence indicating phagocytosis; (J) maximal eFluor 450 staining indicating cell death; and (K) FLICA-1 activation. (L) Culture supernatants were collected and analyzed for IL-1β secretion. Bars represent the mean ± standard error of the mean for at least two independent experiments, each performed in triplicate. Experiments with primary cell experiments include at least three independent donors. Asterisks indicate significance at a <i>p</i>-value of ≤ 0.05 by Tukey’s multiple comparisons post-test for 1-way or 2-way ANOVA, as appropriate.</p

LukAB targets CD11b on human monocytes to potently induce cell death.

<p>(A) THP1 cells were intoxicated with titrations of the indicated purified toxins for 1 hour and LDH release was analyzed. (B) THP1 cells were transduced with either non-targeting shRNA or shRNA against CD11b and surface CD11b levels were evaluated by flow cytometry. (C) THP1 cells described in panel B were intoxicated with titrations of the indicated toxins for 1 hour and LDH release was analyzed. (D) THP1 cells were intoxicated with the indicated concentration of LukAB for 1 hour and analyzed by flow cytometry for permeability to propidium iodide. (E) Primary CD14+ human monocytes were intoxicated with titrations of the indicated toxins for 1 hour and analyzed by flow cytometry for permeability to propidium iodide. EC50 values are also shown. Error bars represent the mean ± standard error of the mean for at least two independent experiments, each performed in triplicate. Primary cell experiments include three independent donors. Asterisks indicate significance at a <i>p</i>-value of ≤ 0.05 by Tukey’s multiple comparisons post-test for 1-way or 2-way ANOVA, as appropriate.</p

Genetic or pharmacologic disruption of Caspase 1 blocks LukAB-induced cytokine secretion but not cell death.

<p>(A) THP1 cells were transfected with siRNA against Caspase 1, ASC or a non-targeting sequence. Cell lysates were collected and analyzed by immunoblot to confirm knockdown of pro-Caspase 1 and ASC. (B) THP1 siRNA cells were incubated with propidium iodide and intoxicated with 50 ng/mL LukAB for 1 hour then analyzed by flow cytometry. (C and D) THP1 siRNA cells were either primed with LTA (500ng/mL) (C) or untreated (D) then intoxicated with LukAB (50ng/mL) for 1 hr before culture supernatants were analyzed for release of IL-1β (C) or IL-18 (D). (E and F) Primary CD14+ human monocytes were incubated with the indicated concentration of z-YVAD-FMK or VX-765 for 30 minutes. Primary monocytes were incubated in the presence of propidium iodide (E) then intoxicated with LukAB (50ng/mL) and analyzed by flow cytometry. (F) Primary monocytes were intoxicated with LukAB (50ng/mL) and culture supernatants were analyzed for release of IL-18. Propidium iodide staining and IL-18 secretion are reported as a fraction of measurement in primary cells not treated with inhibitor. Bars represent the mean ± standard error of the mean for at least two independent experiments, each performed in triplicate. Asterisks indicate significance at a <i>p</i>-value of ≤ 0.05 by Tukey’s multiple comparisons post-test for 1-way or 2-way ANOVA, as appropriate.</p

LukAB is a potent activator of Caspase 1.

<p>(A) THP1 cells were intoxicated with 50 ng/mL LukAB for 1 hour and cell lysates were analyzed by immunoblot for Caspase 1 cleavage, which indicates activation. (B and C) THP1 cells were incubated with FLICA-1 (660-YVAD-FMK) then intoxicated with culture filtrates (1% v/v) from the indicated <i>S</i>. <i>aureus</i> stains. After 1 hour, cells were washed, fixed and analyzed by flow cytometry. Panel B shows a representative flow plot from one experiment, and panel C shows the corresponding mean florescence intensities (MFI). (D) THP1 cells were incubated with FLICA-1, intoxicated with purified LukAB (50 ng/mL) for 1 hour, then washed, fixed and analyzed by flow cytometry. (E) FLICA-1 was added to THP1 cells then LukAB (50 ng/mL) was added for time-course samples in descending order. All samples were washed and fixed at the same time, corresponding to different LukAB incubation times, then analyzed by flow cytometry. THP1 cells (F) and primary CD14+ human monocytes (G) were incubated with FLICA-1 and intoxicated with a dose titration of the indicated purified <i>S</i>. <i>aureus</i> leukotoxins for 1 hour. Cells were washed, fixed and analyzed by flow cytometry. Bars represent the standard error of the mean of triplicate samples. EC50 values are also shown. Each graph is representative of three experiments. Primary cell experiments include three independent donors. Asterisks indicate significance at a <i>p</i>-value of ≤ 0.05 by Tukey’s multiple comparisons post-test for 1-way or 2-way ANOVA, as appropriate.</p

<i>Staphylococcus aureus</i> strains used in this study.

<p>^aNARSA, Network of Antimicrobial Resistance in <i>Staphylococcus aureus</i>.</p><p><i>Staphylococcus aureus</i> strains used in this study.</p

Hla-dependent human T cell and B cell plasma membrane permeablity occurs following 60 minutes exposure to USA300 supernatant.

<p>A) Representative flow cytometry dot plots of human PBMCs stained with propidium iodide following intoxication with filtered 5 hour supernatants from USA300 or USA300Δ<i>hla</i> diluted to a final concentration of 1∶6 for 30 minutes for CD14⁺ PBMCs or 180 minutes for CD3⁺ and CD19⁺ PBMCs. B) Representative histograms of data in part A with CD3⁺, CD14⁺, and CD19⁺ PBMCs exposed to supernatant from USA300 indicated by a dashed line, supernatant from USA300Δ<i>hla</i> indicated with grey shading, and TSB media alone indicated with black shading. Compiled results from at least 4 separate experiments shown in part A using different blood donors for C) CD3⁺, D) CD14⁺ and E) CD19⁺ PBMCs at indicated times following intoxication with *P<0.05, **P<0.01, and ***P<0.001 relative to USA300 as determined by paired one-tailed t-test.</p

Recombinant Hla alone induces programmed cell death in monocytes, T cells and B cells.

<p>Compiled flow cytometry analysis of human PMNs and CD3⁺, CD14⁺, or CD19⁺ PBMCs intoxicated for 60 minutes with increasing concentrations of recombinant Hla then examined using A) Annexin V binding or B) ApoBrdU Tunel assays. Figures represent 3 separate experiments using different blood donors with *P<0.05, **P<0.01, and ***P<0.001 relative to USA300 as determined by one-tailed t-test.</p

Hla induces programmed cell death in human monocytes, T cells and B cells during USA300 infection.

<p>Compiled flow cytometry analysis of human PMNs or PBMCs infected at a MOI of 10 with USA300, USA300Δ<i>hla</i>, USA300Δ<i>hla</i> Comp, USA300Δ<i>saeR/S</i>, or a DPBS control then examined using an Annexin V binding assay at 3 hours (A, C, E, and G) or ApoBrdU Tunel assay at 6 hours (B, D, F, and H). Figures represent 4 separate experiments using different blood donors with *P<0.05, **P<0.01, and ***P<0.001 relative to USA300 as determined by paired one-tailed t-test for A, C, E and G or one-way repeated measures ANOVA with Tukey’s post-test for B, D, F, and H.</p