Polyethylene Glycols Stimulate Ca2+ Signaling, Cytokine Production, and the Formation of Neutrophil Extracellular Traps

Alicja Hinz,1 Sylwia Stankiewicz,1,2 Jacek Jakub Litewka,2,3 Paweł E Ferdek,3 Maja Sochalska,4 Monika Bzowska1 1Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 2Doctoral School of Exact and Natural Sciences Jagiellonian University, Kraków, Poland; 3Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland; 4Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, PolandCorrespondence: Monika Bzowska, Email [email protected]: Given the increased use of polyethylene glycol (PEG) in refining the therapeutic activity of medicines, our research focuses on explaining the potential mechanism of immune reactions associated with this polymer. We aim to investigate the interaction of different types of PEG with mouse and human immune cells, thereby contributing to understanding PEG interactions with the immune system and verifying the proinflammatory activity of the tested polymers.Patients and Methods: Mouse macrophage and neutrophil cell lines, human peripheral blood mononuclear cells, and polymorphonuclear cells isolated from healthy donors were exposed to various PEGs. ROS, NO, and cytokine production were analyzed using fluorescence intensity, absorbance, or cytometric measurements. Toll-like receptor (TLR) signaling was verified using HEK-blue-reporter cell lines. Finally, neutrophil trap formation was studied using immunofluorescence labeling, and calcium imaging was performed using a Ca2+-sensitive indicator and fluorescence microscope.Results: Our findings show that specific PEG and mPEG are not toxic to tested mouse and human cells. However, they exert proinflammatory activity against human immune cells, as evidenced by the increased secretion of proinflammatory cytokines, such as IFN-a2, IFN-γ, TNF-α, MCP-1, IL-8, IL-17A, and IL-23. This phenomenon is independent of PEG signaling via TLR. Additionally, mPEG10 induced the formation of neutrophil extracellular traps and intracellular calcium signaling.Conclusion: Our finding suggests that some PEG types have proinflammatory activity against human immune cells, manifesting in the upregulated production of cytokines and neutrophils trap releasing.Keywords: drug delivery, immunomodulation, in vitro nanotoxicity, nanoparticle functionalization, PEGylated therapeutics