Journal: PLoS ONE

Article Title: Herpes simplex virus type 1 inflammasome activation in proinflammatory human macrophages is dependent on NLRP3, ASC, and caspase-1

doi: 10.1371/journal.pone.0229570

Figure Lengend Snippet: A and B. Primary human MDMs cultured without (M0 A ) or with IFNγ (M1 B ) were incubated with HSV-1 or media for 24 hours. Cell culture supernatants were collected and assayed for IL-18 ( B represents the combination of two experiments). C, D, and E. Primary human MDMs stimulated with IFNγ were cultured in media alone or in media containing 100 μg/mL of VX-765 (Invivogen, San Diego, California) and then incubated with HSV-1, nigericin and LPS (Ng+LPS), or media for 24 hours as outlined in Materials and Methods. Cell culture supernatants were collected and assayed for ( C ) IL-18, ( D ) TNFα, and ( E ) IL-1β. F. MDMs cultured without IFNγ (M0), with IFNγ (M1), or with IFNγ and VX-765 (M1+Vx-765) were infected with HSV-1 for 1 hour followed by citrate wash to inactivate any extracellular virus. Supernatants were collected 24 hours later and plaque forming units (PFU) were determined via standard plaque assay on Vero cells. Data shown are combined from two independent experiments. G. MDMs stimulated with IFNγ (M1) were incubated with HSV-1 or media as well as a neutralizing antibody (WT Ab) and a neutralizing antibody unable to bind to Fc receptors (Fc Silent) for 24 hours. Cell culture supernatants were collected and assayed for IL-18. Differences between groups indicated by brackets were determined by a Student’s t -test. NS, *,**,*** indicate p-values >0.05, <0.05, <0.01, <0.001, respectively. The lower and upper borders of the boxplots represent the 1 st quartile and 3 rd quartile respectively. The median is represented by a horizontal line in the box. The lower and upper whiskers represent 1.5x the interquartile range (IQR) beyond the quartile lines. Each dot represents an individual sample.

Article Snippet: Human IL-18, TNFα, and IL-1β were measured with the human IL-18 ELISA Kit (MBL, Woburn, Massachusetts), human TNFα ELISA Kit (ThermoFisher, Waltham, Massachusetts), and IL-1β MesoScale Discovery (Meso Scale Discovery, Gaithersburg MD) electrochemiluminescence assay according to the manufacturers’ instructions using cell culture supernatant at a 1:5 dilution.

Techniques: Cell Culture, Incubation, Infection, Plaque Assay

Journal: PLoS ONE

Article Title: Herpes simplex virus type 1 inflammasome activation in proinflammatory human macrophages is dependent on NLRP3, ASC, and caspase-1

doi: 10.1371/journal.pone.0229570

Figure Lengend Snippet: A. THP-1 cells were stimulated overnight with PMA (100 ng/mL) and then incubated with HSV-1, nigericin and LPS (Ng+LPS), or media, as outlined in Materials and Methods, for 24 hours. Cell culture supernatants were collected and IL-18 was measured via ELISA. B. THP-1 cells with the indicated gene disrupted via CRISPR-cas9 (Δ) were stimulated overnight with PMA, infected with HSV-1 for 1 hour followed by citrate wash to inactivate any extracellular virus. Supernatants were collected 24 hours later and PFU were determined via standard plaque assay on Vero cells. Data shown are combined from two independent experiments. C. Cell lysates from THP-1 cells infected with HSV-1 or mock infected were probed for caspase-1 or β-actin via western blot. D. THP-1 cells with the indicated gene disrupted via CRISPR-cas9 (Δ) were stimulated overnight with PMA and then incubated with HSV-1, nigericin and LPS (Ng+LPS), or media for 24 hours before IL-18 was measured in cell supernatants. ΔHUMCYC cells are labeled as “WT.” Differences between groups indicated by brackets were determined by a Student’s t -test. NS, *,**,*** indicate p-values >0.05, <0.05, <0.01, <0.001, respectively.

Article Snippet: Human IL-18, TNFα, and IL-1β were measured with the human IL-18 ELISA Kit (MBL, Woburn, Massachusetts), human TNFα ELISA Kit (ThermoFisher, Waltham, Massachusetts), and IL-1β MesoScale Discovery (Meso Scale Discovery, Gaithersburg MD) electrochemiluminescence assay according to the manufacturers’ instructions using cell culture supernatant at a 1:5 dilution.

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, CRISPR, Infection, Plaque Assay, Western Blot, Labeling

Journal: PLoS ONE

Article Title: Herpes simplex virus type 1 inflammasome activation in proinflammatory human macrophages is dependent on NLRP3, ASC, and caspase-1

doi: 10.1371/journal.pone.0229570

Figure Lengend Snippet: A and B. Primary human MDMs cultured without (M0 A ) or with IFNγ (M1 B ) were incubated with HSV-1, UV irradiated HSV-1 (HSV-1/UV), or media for 24 hours. Cell culture supernatants were collected and assayed for IL-18. C. Primary human MDMs cultured with IFNγ were incubated with HSV-1, UV irradiated HSV-1 (HSV-1/UV), or media for 4 hours and 8 hours. Cell culture supernatants were collected and assayed for IL-18. Differences between indicated conditions within a cell type were determined by a one-way ANOVA with Tukey HSD post-hoc analysis. NS, *,**,*** indicate p-values >0.05, <0.05, <0.01, <0.001 respectively.

Article Snippet: Human IL-18, TNFα, and IL-1β were measured with the human IL-18 ELISA Kit (MBL, Woburn, Massachusetts), human TNFα ELISA Kit (ThermoFisher, Waltham, Massachusetts), and IL-1β MesoScale Discovery (Meso Scale Discovery, Gaithersburg MD) electrochemiluminescence assay according to the manufacturers’ instructions using cell culture supernatant at a 1:5 dilution.

Techniques: Cell Culture, Incubation, Irradiation

Journal: PLoS ONE

Article Title: Herpes simplex virus type 1 inflammasome activation in proinflammatory human macrophages is dependent on NLRP3, ASC, and caspase-1

doi: 10.1371/journal.pone.0229570

Figure Lengend Snippet: A. THP-1 cells were stimulated overnight with PMA (5 ng/mL) and then incubated with HSV-1, UV irradiated HSV-1 (HSV-1/UV), or media for 24 hours. Cell culture supernatants were collected and IL-18 was measured via ELISA. B. THP-1 cells were stimulated with PMA (5 ng/mL) and then with IFN0γ (25 ng/mL) the following day for 24 hours prior to incubation with HSV-1, UV irradiated HSV-1 (HSV-1/UV), or media alone for 24 hours. Cell culture supernatants were collected and IL-18 was measured via ELISA. ΔHUMCYC cells are labeled as “WT.” Differences between indicated conditions within a cell type were determined by a one-way ANOVA with Tukey HSD post-hoc analysis. NS, *,**,*** indicate p-values >0.05, <0.05, <0.01, <0.001 respectively.

Article Snippet: Human IL-18, TNFα, and IL-1β were measured with the human IL-18 ELISA Kit (MBL, Woburn, Massachusetts), human TNFα ELISA Kit (ThermoFisher, Waltham, Massachusetts), and IL-1β MesoScale Discovery (Meso Scale Discovery, Gaithersburg MD) electrochemiluminescence assay according to the manufacturers’ instructions using cell culture supernatant at a 1:5 dilution.

Techniques: Incubation, Irradiation, Cell Culture, Enzyme-linked Immunosorbent Assay, Labeling