Role of Ambient Gas Composition on Cold Physical Plasma-Elicited Cell Signaling in Keratinocytes.

Article Role of Ambient Gas Composition on Cold Physical Plasma-Elicited Cell Signaling in Keratinocytes Anke Schmidt,1,* Sander Bekeschus,2 Helena Jablonowski,2 Annemarie Barton,2 Klaus-Dieter Weltmann,1,2 and Kristian Wende2 1 Plasma Life Science, Leibniz Institute for Plasma Science and Technology (INP Greifswald), Greifswald, Germany; and 2Center for Innovation Competence (ZIK) Plasmatis, Greifswald, Germany ABSTRACT A particularly promising medical application of cold physical plasma is the support of wound healing. This is presumably achieved by modulating inflammation as well as skin cell signaling and migration. Plasma-derived reactive oxygen and nitrogen species (ROS/RNS) are assumed the central biologically active plasma components. We hypothesized that modulating the environmental plasma conditions from pure nitrogen (N2) to pure oxygen (O2) in an atmospheric pressure argon plasma jet (kINPen) will change type and concentration of ROS/RNS and effectively tune the behavior of human skin cells. To investigate this, HaCaT keratinocytes were studied in vitro with regard to cell metabolism, viability, growth, gene expression signature, and cytokine secretion. Flow cytometry demonstrated only slight effects on cytotoxicity. O2 shielding provided stronger apoptotic effects trough caspase-3 activation compared to N2 shielding. Gene array technology revealed induction of signaling and communication proteins such as immunomodulatory interleukin 6 as well as antioxidative and proproliferative molecules (HMOX1, VEGFA, HBEGF, CSF2, and MAPK) in response to different plasma shielding gas compositions. Cell response was correlated to reactive species: oxygen-shielding plasma induces a cell response more efficiently despite an apparent decrease of hydrogen peroxide (H2O2), which was previously shown to be a major player in plasma-cell regulation, emphasizing the role of non-H2O2 ROS like singlet oxygen. Our results suggest differential effects of ROS- and RNS-rich plasma, and may have a role in optimizing clinical plasma applications in chronic wounds. INTRODUCTION In the last few years, cold physical plasma gained attention as a promising tool for medical applications in wound management and care. It is well known that plasma is highly antimicrobial against prokaryotes and biofilms in vitro (1–5) or antiseptic regarding wounds in vivo (6–8). Plasma therapy seems promising because it is low in temperature, nonmutagenic, and well tolerated by tissues, and with a wide range of sources, easy to use (9–11). The first preclinical (12) as well as clinical studies (13,14) fostered the in vivo potential of plasma treatment of poorly or nonhealing wounds in patients. Cold plasma is defined as a partially ionized gas producing reactive oxygen (ROS) and nitrogen species (RNS), electrons, ions, an electric field, and ultraviolet, thermal, and infrared radiation (15). The kINPen plasma jet is generally operated with pure argon. By admixing molecular gases, the composition of reactive species in the plasma Submitted February 16, 2017, and accepted for publication April 24, 2017. *Correspondence: Editor: Catherine Galbraith. http://dx.doi.org/10.1016/j.bpj.2017.04.030 effluent can be modulated. Addition of N2 leads to increased generation of RNS (16), whereas addition of O2 shifts the balance more toward the ROS (17). Feed gas humidity is an additional source of ROS production (18). The effluents species composition is also affected by the plasma jet’s surroundings as components of the ambient air diffuse into the plasma effluent and alter yields of ROS and RNS (19). Biological systems such as cells and tissues have established various mechanisms, enabling them to control the amount of reactive species to stabilize a baseline level of reactive oxygen intermediates that is tolerable and partly even beneficial to the cell (20,21). Therefore, a more balanced concept of the quantity and the effects of potentially dangerous particles such as ROS/RNS on living cells is accepted (22) and is called ‘‘hormesis’’ (23,24). Subtle increase in ROS/RNS has even been associated with increased lifespan (25), as opposed to the implications of the freeradical theory of aging. Plasma-generated reactive species are believed to play a central role in the medical utilizations of plasma (26), as they govern a variety of processes such as distinct cytotoxicity (27,28) and cell signaling (29,30). Ó 2017 Biophysical Society. Biophysical Journal 112, 2397–2407, June 6, 2017 2397 Schmidt et al. Consequently, redox-related signaling plays an important role in cellular reactions to plasma exposure. Keratinocytes constitute the uppermost layers of the skin and as such are central players of the reepithelialization during wound repair by migrating and proliferating onto the provisional matrix of the underlying granulation tissue (31). Using HaCaT keratinocytes as an in vitro cell culture system, we here investigated plasma-derived activation of skin cells under different controlled conditions. With regard to this, shielding of an unchanged argon gas plasma created by the kINPen was varied from pure nitrogen to pure oxygen. After exposure of plasma-treated medium to HaCaT cells, mapping of the gene expression signature was conducted to obtain deeper insights into the underlying mechanisms and molecular biological pathways. We identified alterations in cell viability, gene and protein expression, and cytokine release. MATERIALS AND METHODS Cell culture and plasma treatment All experiments were performed using human adult low-calcium high-temperature keratinocytes HaCaT (DKFZ, Heidelberg, Germany) cultivated in Roswell Park Memorial Institute 1640 medium supplemented with 8% fetal bovine serum (Sigma-Aldrich, Munich, Germany), 2 mM L-glutamine, 0.1 mg/L streptomycin, and 100 U/mL penicillin (Lonza, Visp, Switzerland). Plasma treatment was performed using the atmospheric pressure plasma jet kINPen 09 (voltage of 2–6 kVpp, frequency
1 MHz; Neoplas, Greifswald, Germany), which was surrounded by a gas-shielding device, separating the plasma effluent from the ambient air. The plasma was ignited 1 h before the treatment to remove humidity from the tubing. The kINPen was operated with argon (three standard liters per min, slm), and the shielding gas consisted of different ratios of O2 and N2 with a total volumetric flow rate of 5 slm. The oxygen/nitrogen (in %) in the shielding gas was varied in five steps (0:100; 25:75; 50:50; 75:25; 100:0). The parameters for each shielding gas composition were adjusted 3 min before treatment to guarantee constant conditions and homogeneous gas mixtures. The plasma treatment of 1,000,000 cells was performed in an indirect way: 5 mL of cell culture medium were treated in a 60-mm petri dish in a meandering way for desired time at a fixed distance of 12 mm. Subsequently, the cell medium was replaced by plasma-treated medium. Positive control cells received 100 mM H2O2. In previous wor (...truncated)