Differential Effect of Non-Thermal Plasma RONS on Two Human Leukemic Cell Populations

Abstract

Non-thermal plasma application to cancer cells is known to induce oxidative stress, cytotoxicity and indirect immunostimulatory effects on antigen presenting cells (APCs). The purpose of this study was to evaluate the responses of two leukemic cell lines-Jurkat T lymphocytes and THP-1 monocytes-to NTP-generated reactive oxygen and nitrogen species (RONS). Both cell types depleted hydrogen peroxide, but THP-1 cells neutralized it almost immediately. Jurkat cells transiently blunted the frequency-dependent increase in nitrite concentrations in contrast to THP-1 cells, which exhibited no immediate effect. A direct relationship between frequency-dependent cytotoxicity and mitochondrial superoxide was observed only in Jurkat cells. Jurkat cells were very responsive to NTP in their display of calreticulin and heat shock proteins 70 and 90. In contrast, THP-1 cells were minimally responsive or unresponsive. Despite no NTP-dependent decrease in cell surface display of CD47 in either cell line, both cell types induced migration of and phagocytosis by APCs. Our results demonstrate that cells modulate the RONS-mediated changes in liquid chemistry, and, importantly, the resultant immunomodulatory effects of NTP can be independent of NTP-induced cytotoxicity.

Keywords: calreticulin; damage-associated molecular patterns (DAMPs); heat shock proteins; hydrogen peroxide; immunotherapy; nitrite; oxidative stress; phagocytosis; plasma medicine.

Figure 1

Experimental set-up for exposure of RPMI, Jurkat cells, or THP-1 cells to NTP. A nanosecond-pulsed dielectric barrier discharge (DBD) device was used to deliver NTP to cells or RPMI aliquoted into a 24-well plate. Plasma generation frequency (30–105 Hz) was controlled through the function generator, which was set to deliver a 10 s exposure time. NTP was delivered to cells using a dielectric-encased electrode positioned by a Z-positioner over media in the absence or presence of cells at a distance of 2 mm from the bottom of the well.

Figure 2

Jurkat or THP-1 cells alter the RONS composition of NTP-exposed media. (a) Hydrogen peroxide concentration in RPMI in the absence of cells or containing Jurkat or THP-1 cells and (b) nitrite concentration in RPMI without cells or containing Jurkat or THP-1 cells immediately (T0—red) and 24 h (T24—green) after NTP exposure. (c) Hydrogen peroxide concentration is lower in the presence of Jurkat or THP-1 cells both at 30 Hz and 105 Hz at T0 and T24 after NTP exposure. (d) NTP exposure of RPMI in the absence of cells results in a higher concentration of nitrite in RPMI in the absence of cells than with Jurkat cells, immediately following 105 Hz exposure. Data are presented as mean ± SD from one experiment (n = 3). Significance was calculated using an unpaired Student’s t-test (** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 3

NTP induces cytotoxicity in Jurkat cells but not in THP-1 in a frequency-dependent manner up to 105 Hz. (a) Viability of Jurkat and THP-1 cells 24 h post NTP exposure. (b) Mitochondrial superoxide production increased in both Jurkat and THP-1 cells 24 h post-NTP exposure, measured both as percent MitoSOX-positive cells and overall mean fluorescence intensity (MFI). Dotted line indicates no change with respect to cells not exposed to NTP. Data are presented as mean ± SEM. Significance was calculated using Brown–Forsythe one-way ANOVA and Dunnett’s post-hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 4

Jurkat and THP-1 cells display pro-phagocytic DAMPs following NTP exposure. The magnitude of increased surface display of (a) CRT, (b) HSP70, and (c) HSP90 differs between NTP-exposed Jurkat and THP-1 cells 24 h post-exposure. Dotted line indicates no change with respect to cells in the absence of NTP. Data are presented as mean ± SEM. Significance was calculated using a Kruskal–Wallis test with Dunnett’s post-hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 5

CD47 expression on Jurkat or THP-1 cells is not altered immediately or 24 h post NTP exposure. CD47 expression on Jurkat and THP-1 cells is comparably displayed at both (a) 0 h (T0) and (b) 24 h (T24) after NTP exposure relative to cells in the absence of NTP. Dotted line indicates no change with respect to cells not exposed to NTP. Data are presented as mean ± SEM. Significance was calculated using a Kruskal–Wallis test with Dunnett’s post-hoc test.

Figure 6

NTP-exposed Jurkat and THP-1 monocytes are phagocytosed by macrophages. Percentage of THP-1 macrophages completing phagocytosis (WGA+HCST+) of Jurkat or THP-1 cells. Dotted line indicates no change with respect to cells in the absence of NTP. Data are presented as mean ± SEM. Significance was calculated using a Kruskal–Wallis test with Dunnett’s post-hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 7

THP-1 monocyte migration is stimulated in response to NTP-exposed cells. The number of migrated (CTFR+) monocytes was increased 24 h post co-culture by NTP-exposed Jurkat or THP-1 cells. Data are presented as mean ± SEM. Significance was calculated using a Kruskal–Wallis test with Dunnett’s post-hoc test (* p < 0.05, ** p < 0.01, **** p < 0.0001).