Argon Plasma Exposure Augments Costimulatory Ligands and Cytokine Release in Human Monocyte-Derived Dendritic Cells

Abstract

Cold physical plasma is a partially ionized gas expelling many reactive oxygen and nitrogen species (ROS/RNS). Several plasma devices have been licensed for medical use in dermatology, and recent experimental studies suggest their putative role in cancer treatment. In cancer therapies with an immunological dimension, successful antigen presentation and inflammation modulation is a key hallmark to elicit antitumor immunity. Dendritic cells (DCs) are critical for this task. However, the inflammatory consequences of DCs following plasma exposure are unknown. To this end, human monocyte-derived DCs (moDCs) were expanded from isolated human primary monocytes; exposed to plasma; and their metabolic activity, surface marker expression, and cytokine profiles were analyzed. As controls, hydrogen peroxide, hypochlorous acid, and peroxynitrite were used. Among all types of ROS/RNS-mediated treatments, plasma exposure exerted the most notable increase of activation markers at 24 h such as CD25, CD40, and CD83 known to be crucial for T cell costimulation. Moreover, the treatments increased interleukin (IL)-1α, IL-6, and IL-23. Altogether, this study suggests plasma treatment augmenting costimulatory ligand and cytokine expression in human moDCs, which might exert beneficial effects in the tumor microenvironment.

Keywords: CAP; RNS; ROS; cancer; cold atmospheric pressure plasma; hydrogen peroxide; hypochlorous acid; moDCs; peroxynitrite; reactive oxygen and nitrogen species.

Figure A1

Standard curves from the multiplex cytokine analysis for each analyte across a range of concentrations shown together with the corresponding average mean fluorescence intensities (Avg(MFI)). All curve fittings had an R² of greater 0.99, providing great accuracy for the quantification of absolute cytokine levels in moDC culture supernatants.

Figure 1

Study protocol and toxicity comparison. (a) Image of argon plasma treatment of cells in 24-well plates; (b) scheme of study protocol; (c,d) overlay 4′,6-diamidino-2-phenylindole (DAPI) histograms of monocyte-derived DCs (moDCs) and lymphocytes according to the indicated treatments (c) and quantification of the percentage of viable moDCs and lymphocytes (d) treated together in a single well. Data are mean and standard error of three experiments, and statistical analysis was performed using t-test with p < 0.001 (***) differing significantly or non-significantly (ns).

Figure 2

Argon plasma treatment and reactive oxygen and nitrogen species (ROS/RNS) have dose-dependent toxicity profiles. (a) Kinetic metabolic activity of moDCs treated as indicated over 6 h; (b–f) metabolic activity in response to several argon plasma treatments times or concentrations of ROS/RNS or LPS at 24 h; (g–k) viability in response to several argon plasma treatment times or concentrations of ROS/RNS or lipopolysaccharide (LPS) at 24 h; and (l) heatmap for comparison between reduction in metabolic activity and viability. Data are mean and standard error of 3–6 different donors.

Figure 3

Argon plasma treatment and ROS/RNS modulate the surface marker expression profiles. (a) overlay histograms of several cell surface markers of unstained, stained, stained, and LPS-pulsed human moDCs; (b) quantification and fold-change differences in human moDCs treated as indicated and analyzed 24 h later; (c) data summary using principal-component analysis. Data are mean and standard error of 3–6 different donors. Statistical analysis was done using one-way analysis of variances with p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Figure 4

Argon plasma treatment and ROS/RNS modulate the cytokine release profiles. Cells were treated as indicated, supernatants were collected 24 h later, and absolute cytokine concentrations of 10 analytes (a–j) were assessed. All data were also related to each other using principal component analysis (k). Data are violin plots and median (red lines) of 3–6 different donors. Statistical analysis was done using one-way analysis of variances with p < 0.05 (*). Dashed lines show values of LPS-positive controls of moDCs included for the analytes.