IFN-γ in supernatants of IL-23 plus IL-1β stimulated CD56⁺ cells primes monocytes for IL-12p70 production in response to LPS.

<p>1 ·10⁵ CD14⁺ monocytes were stimulated with 100 ng/ml LPS in combination with 2.5 ng/ml IFN-γ, supernatants of unstimulated CD56⁺ cells, supernatants of IL-23 plus IL-1β stimulated CD56⁺ cells, or medium alone for 24 hours (A). 1 ·10⁵ CD14⁺ monocytes were stimulated with 100 ng/ml LPS in combination supernatants of IL-23 plus IL-1β stimulated CD56+ cells, plus or minus 2 µg/ml anti-IFN-γ for 24 hours (B). Supernatants were taken and IL-12p70 production was measured by ELISA. Data are means±SD of triplicates from one representative experiment of three.</p

GM-CSF and IFN-γ prime monocytes for enhanced IL-23 production.

<p>1 ·10⁵ CD14⁺ monocytes were prestimulated with GM-CSF (A) or IFN-γ (B) for 16 hours in concentrations indicated. Subsequently cells were stimulated with heat killed Salmonella (moi = 10) or left unstimulated for 24 hours. Supernatants were taken and IL-23 production was measured by ELISA. Data are means±SD of triplicates in one representative experiment of five.</p

Supernatants of <i>Salmonella</i>-infected Mφ1 induce IFN-γ production in primary human CD56⁺ cells.

<p>(A) IFN-γ production by CD56⁺ NK/NK-like T cells from two donors. Cells were cultured for 48 hours in the presence of supernatants obtained from uninfected or <i>Salmonella</i> infected Mφ1 (from two donors) with or without additional exogenous recombinant IL-18. As a control, medium only was used. Data are means±SD of triplicates in one representative experiment of three. (B) IFN-γ production in CD56⁺ cells induced by supernatants of <i>Salmonella</i>-infected Mφ1 is blocked by neutralizing IL-12p40 or IL-1β, but not by neutralizing IL-18. CD56⁺ NK/NK-like T cells were cultured for 48 hours in the presence or absence of supernatant obtained from Mφ1 cells infected with <i>Salmonella</i>, in the presence or absence of an IL-12p40, IL-18 or IL-1β neutralizing antibody. IFN-γ concentration was determined by ELISA. IFN-γ production induced by supernatants of <i>Salmonella</i> infected Mφ1 is set at 100%. A paired two-tailed student's t-test was used for statistical analysis. Data are means±SEM of experiments with cells obtained from 10 different donors.</p

IL-23 in combination with IL-1β or IL-18 induces IFN-γ production in both CD56⁺/CD3⁻ NK cells and CD56⁺/CD3⁺ NK-like T cells.

<p>Anti-CD56 MACS bead isolated cells were rested overnight and then left unstimulated or stimulated with 10 ng/ml IL-1β, 100 ng/ml IL-18, 10 ng/ml IL-23, 10 ng/ml IL-1β plus 10 ng/ml IL-23 or 100 ng/ml IL-18 plus 10 ng/ml IL-23, for 48 hours. Cells were fixed, permeabilised and labeled with anti-human CD3-PE, anti-human CD56-FITC and anti-human IFN-γ-Alexa 647. Unstimulated cells and cells stimulated with a single cytokine do not produce significant amounts of IFN-γ. CD3⁻ NK cells stimulated with IL-1β or IL-18 in combination with IL-23 induce significant IFN-γ production, while in CD3⁺ NK-like Tcells only IL-23 plus IL-18 induces significant IFN-γ production. Data are means±SD of experiments with cells obtained from 8 donors. Two-tailed paired t-tests were performed of stimulated cells versus unstimulated cells. * indicates a p-value<0.05.</p

Percentages of IFN-γ producing CD56⁺ cells after stimulation with cytokines.

<p>Percentages of IFN-γ producing CD56⁺/CD3⁻ NK cells and CD56⁺/CD3⁺ NK-like T cells for each of eight donors after stimulation of CD56⁺ cells with the indicated cytokines for 48 h.</p

TLR stimuli and <i>Salmonella</i> infection induce IL-23 production in human monocytes and Mφ1.

<p>1 ·10⁵ overnight rested anti-CD14 beads-isolated monocytes (A) or 5 ·10⁴ Mφ1 (B) were stimulated with 100 ng/ml LPS, 200 ng/ml flagellin (flag), 1 µg/ml Pam3CSK4, 100 µg/ml Zymosan A, 1 µg/ml CL075, 1 µg/ml CL087, 100 ng/ml LPS plus 200 ng/ml flagellin, LPS 100 ng/ml plus 1 µg/ml CL075 for 24 hours, or left unstimulated. (C) Mφ1 were infected with live <i>Salmonella</i> with a multiplicity of infection (MOI) of 10, left uninfected or were stimulated with 100 ng/ml LPS. Supernatants were collected after 24 hours and IL-23 protein production was assessed by ELISA. Data are means±standard deviation (SD) of triplicates in one representative experiment of five (A and B) or means of duplicates in one representative experiment of three (C).</p

IL-23, in combination with IL-1β, induces IFN-γ production in primary human CD56⁺ cells.

<p>Anti-CD56 MACS bead isolated cells were rested overnight and then left unstimulated or stimulated with indicated concentrations of IL-23 in combination with various concentrations of IL-1β. Supernatants were collected 24 hours after stimulation and IFN-γ concentration was measured by ELISA. IL-23 and IL-1β synergize in the induction of IFN-γ. One representative experiment of three is shown.</p

<i>Salmonella</i> infection induces IL-18 and IL-1β production in Mφ1.

<p>3.3 ·10⁵ Mφ1 of five different donors were left unstimulated, stimulated with LPS or infected with <i>Salmonella</i> with a MOI of 10. Supernatants were collected after 24 hours and IL-18 (A) and IL-1β (B) production was measured by ELISA. Each dot represents one donor. The mean is indicated by a horizontal line. A paired two-tailed student's t-test was used for statistical analysis. n.d. = not detected.</p