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Schindler et al. Microparticles in neuroimmune signaling

onto 96-well plates for optical density measurement at THP-1 cells that had been stimulated with IFN-γ alone
570 nm using a microplate reader. The cell viability was or IFN-γ plus LPS were added to THP-1 cell cultures.
expressed as a percent of the value obtained from cells At the concentration tested, none of the isolated MPs
treated with medium only. significantly affected THP-1 cell viability [Figure 2A].
Cell-free supernatants from the THP-1 cells treated
ELISA with different types of MPs were transferred onto SH-
Concentrations of IL-6, TNF-α, and MCP-1 in cell-free SY5Y neuroblastoma cells to determine secretion of
supernatants from THP-1 cells stimulated for 48 h with cytotoxins by MP-stimulated THP-1 cells [Figure 2B].
THP-1-derived MPs were measured using Peprotech The MPs isolated from unstimulated THP-1 cells as well
ELISA development kits according to the manufacturer’s as those isolated from IFN-γ-stimulated THP-1 cells, did
instructions. The detection limits for the IL-6, TNF-α, and not induce THP-1 cell toxicity towards SH-SY5Y cells.
MCP-1 ELISAs were experimentally determined to be However, the MPs isolated from THP-1 cells stimulated
0.007 ng/mL, 0.09 ng/mL, and 1.02 ng/mL, respectively. with IFN-γ plus LPS induced cytotoxicity of THP-1
cells, resulting in a statistically significant decrease in
Data analyses viability of the neuronal cells compared to SH-SY5Y
Statistical analyses of the data were conducted using cells exposed to supernatants from unstimulated
SPSS software (version 22.0, IBM SPSS, Chicago THP-1 cells (P = 0.013). The THP-1 cell toxicity induced
IL, USA) and GraphPad PRISM software (version by the MP population from IFN-γ plus LPS-stimulated
6.0, GraphPad Software Inc, La Jolla CA, USA). THP-1 donor cells was also significantly different
Randomized blocks design analysis of variance from the effect of the supernatants from THP-1 cells
(ANOVA) followed by Fisher’s least-significant difference stimulated with MPs isolated from unstimulated THP-1
(LSD) post hoc test was used to determine significance donor cells (P = 0.001) and THP-1 cells stimulated with
of findings. Data are presented as means ± standard IFN-γ alone (P = 0.048).
error of the mean (S.E.M.). Statistical significance
was considered at a P-value less than 0.05. Data from IFN-γ enhances THP-1 cell cytotoxicity induced
3-11 independent experiments for each figure are by stimulated THP-1 cell-derived MPs
presented. The synergistic effect of IFN-γ and THP-1 cell-derived
MPs on THP-1 cell cytotoxic secretions was also
RESULTS studied [Figure 3A]. The combination of IFN-γ and
IFN-γ plus LPS-stimulated THP-1 cell-derived MPs
MP release by stimulated human monocytic was significantly toxic towards THP-1 cells compared
THP-1 cells to THP-1 cells treated with vehicle solution alone
The ability of various stimuli to trigger MP release by (P = 0.0001), IFN-γ alone (P = 0.002), and MPs isolated
monocytic THP-1 cells was investigated. THP-1 cells from IFN-γ plus LPS-stimulated THP-1 cells alone (P =
were stimulated for 24 h with IFN-γ or a combination 0.0001). Supernatants from the differentially treated
of IFN-γ plus LPS, which has previously been shown THP-1 cells were transferred onto SH-SY5Y cells
to induce maximal stimulation of these cells. THP-1 cell and their viability was assessed after 72 h incubation.
[38]
supernatants were collected and MPs isolated using Figure 3B illustrates that the cytotoxicity of MPs derived
differential centrifugation. Representative scatter plots from IFN-γ plus LPS-stimulated THP-1 donor cells was
shown on Figure 1A-C illustrate that MPs were released enhanced by IFN-γ being present during the incubation
by unstimulated and stimulated THP-1 cells as shown of THP-1 cells with these MPs.
by the Annexin-V positive events within the MP size
gate. The number of MPs released per million THP-1 MPs derived from TFAM plus IFN-γ-stimulated
cells was calculated. Figure 1D shows that significantly THP-1 cells induce cytotoxicity of THP-1 cells
more MPs were released by THP-1 cells stimulated The ability of MPs derived from TFAM-stimulated
with a combination of IFN-γ plus LPS compared to cells monocytic THP-1 cells to induce cytotoxic secretions
stimulated with IFN-γ alone (P = 0.001) or unstimulated from THP-1 cells was also investigated. MTT assay
cells (P = 0.001). showed no toxicity of the isolated MPs towards THP-1
cells at the concentration tested [Figure 4A]. Treatment
Effects of THP-1 cell-derived MPs on THP-1 of THP-1 cells with MPs isolated from TFAM-stimulated
monocytic cell viability and their toxic secretions THP-1 donor cells did not cause secretion of cytotoxins
Next, we studied whether MPs derived from by THP-1 cells. However, MPs from THP-1 cells
unstimulated and stimulated THP-1 cells induce stimulated with TFAM in combination with IFN-γ
cytotoxicity of THP-1 cells in a paracrine fashion. MPs induced THP-1 cell cytotoxicity resulting in a statistically
derived from unstimulated THP-1 cells (control) or significant decrease in SH-SY5Y cell viability compared
Neuroimmunology and Neuroinflammation ¦ Volume 3 ¦ October 28, 2016 235