## 4 Discussion  
Monocytes and macrophages are important regulators of inflammation. As these cells are both a source and target of ROS, this study strived to investigate how a mixture of exogenous ROS--generated with a novel medical technology--affected inflammatory and phenotypic traits important for monocyte-to-macrophage differentiation. Our data suggested that THP-1 monocytes showed cellular differentiation in response to cold physical plasma-derived ROS. This was reflected by the surface marker profile in response to plasma. CD55 was reported before to be important in the differentiation of myeloid cells [48]. The largest increase was seen with CD271 (p75 NGF receptor) that is frequently used as a stem cell marker [49; 50; 51; 52] and a target in melanoma treatment [53]. It was previously found to be upregulated in primary monocytes/macrophages deprived of endogenously produced NGF after LPS stimulation [54]. Further, an increase of HLA-ABC was seen in plasma-treated THP-1. Upregulation of this molecule is associated with mature dendritic cells [55], mature macrophages [56], and monocyte-to-macrophage differentiation upon GM-CSF and IL4 stimuli [57] independent of the M1 or M2 phenotype [58]. This study also found CD11c, a marker for mature monocytes and macrophages [59; 60], to be upregulated in plasma-treated THP-1 cells. Likewise, CD69 was identified as an activation marker in U-937 monocytes in response to LPS [61]. Using primary monocytes from healthy human donors, a specific pattern of ROS-modulated surface marker expression was found that differed from that of THP-1 monocytes. The origin of the THP-1 cell line, a commonly used model for immune modulation, is the blood of a patient with acute monozygotic leukemia [62]. Apart from major similarities, both cells have distinct characteristics. THP-1 cells are immortal, (in contrast to primary monocytes) highly proliferative, and have higher intrinsic phagocytic capacities [63]. Naturally, gene expression [63], surface markers [64], as well as the sensitivity towards LPS [65] and the associated cytokine response [66] varies between THP-1 cells  
Figure 6: Co-culture of plasma-stimulated THP-1 monocytes with human A375 melanoma cells. **(a)** The representative images of the time course of co-culture with 120 s plasma-treated THP-1 cells up to 8 h indicated by t, green arrows point to myeloid cells, red arrows in t = 1 to melanoma cells; **(b)** The quantification of DAPI as dead-cell dye in cell trace red (CTR) labeled melanoma cells at 8 h after initiation of co-culture. **(b)** The quantification of DAPI uptake in melanoma cells in untreated, or 20 s or 120 s plasma-treated conditions (\(\pm\)PMA). The data are the mean + SD from two independent experiments with three technical replicates each. The statistical analysis was performed using one-way anova with Dunnett post-testing to respective controls. The seconds (s) indicate plasma treatment time, ctrl = control, scale bar = 50 \(\upmu\)m.  
and primary monocytes. These factors could be responsible for the slightly different ROS-induced differentiation patterns observed in both cell types, despite the fact that the treatment varied by a factor of 6 to 12, owing to the increased robustness of THP-1 cells towards ROS-induced cell death [67]. In primary monocytes, the authors observed an increase of CD80 being a stimulatory receptor for T cell activation [68] and CD163, a receptor for the hemoglobin-haptoglobin complex and M2 macrophage marker [68] with plasma treatment. The mannose receptor and M2 macrophage marker CD206 was not changed in plasma treated primary monocytes, partly arguing against such M2 phenotype upon ROS-induced differentiation. This view is supported by an increase in HLA-DR expression (an M1 macrophage marker) [69] in plasma-treated primary monocytes and counteracted by an increase of fibronectin (an M2 macrophage marker) [70]. Hence, plasma-derived ROS were able to induce monocyte-to-macrophage maturation but did not favor a specific phenotype in these cells.