The Ca2+ concentration impacts the cytokine production of mouse and human lymphoid cells and the polarization of human macrophages in vitro

PLOS ONE RESEARCH ARTICLE The Ca2+ concentration impacts the cytokine production of mouse and human lymphoid cells and the polarization of human macrophages in vitro Yusuf Cem Eskiocak1☯, Zeynep Ozge Ayyildiz1,2☯, Sinem Gunalp1,2‡, Asli Korkmaz ID1,2‡, Derya Goksu Helvaci ID3‡, Yavuz Dogan4, Duygu Sag1,2,5, Gerhard Wingender ID1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Izmir Biomedicine and Genome Center (IBG), Balcova/Izmir, Turkey, 2 Department of Genome Sciences and Molecular Biotechnology, Izmir International Biomedicine and Genome Institute, Dokuz Eylul University, Balcova/Izmir, Turkey, 3 School of Medicine, Dokuz Eylul University, Balcova/Izmir, Turkey, 4 Department of Microbiology, Faculty of Medicine, Dokuz Eylul University, Balcova/Izmir, Turkey, 5 Department of Medical Biology, Faculty of Medicine, Dokuz Eylul University, Balcova/Izmir, Turkey ☯ These authors contributed equally to this work. ‡ SG, AK and DGH also contributed equally to this work. * OPEN ACCESS Citation: Eskiocak YC, Ayyildiz ZO, Gunalp S, Korkmaz A, Helvaci DG, Dogan Y, et al. (2023) The Ca2+ concentration impacts the cytokine production of mouse and human lymphoid cells and the polarization of human macrophages in vitro. PLoS ONE 18(2): e0282037. https://doi.org/ 10.1371/journal.pone.0282037 Editor: Nazmul Haque, TotiCell Limited, Bangladesh, BANGLADESH Received: May 25, 2022 Accepted: February 6, 2023 Published: February 24, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0282037 Copyright: © 2023 Eskiocak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Abstract Various aspects of the in vitro culture conditions can impact the functional response of immune cells. For example, it was shown that a Ca2+ concentration of at least 1.5 mM during in vitro stimulation is needed for optimal cytokine production by conventional αβ T cells. Here we extend these findings by showing that also unconventional T cells (invariant Natural Killer T cells, mucosal-associated invariant T cells, γδ T cells), as well as B cells, show an increased cytokine response following in vitro stimulation in the presence of elevated Ca2+ concentrations. This effect appeared more pronounced with mouse than with human lymphoid cells and did not influence their survival. A similarly increased cytokine response due to elevated Ca2+ levels was observed with primary human monocytes. In contrast, primary human monocyte-derived macrophages, either unpolarized (M0) or polarized into M1 or M2 macrophages, displayed increased cell death in the presence of elevated Ca2+ concentrations. Furthermore, elevated Ca2+ concentrations promoted phenotypic M1 differentiation by increasing M1 markers on M1 and M2 macrophages and decreasing M2 markers on M2 macrophages. However, the cytokine production of macrophages, again in contrast to the lymphoid cells, was unaltered by the Ca2+ concentration. In summary, our data demonstrate that the Ca2+ concentration during in vitro cultures is an important variable to be considered for functional experiments and that elevated Ca2+ levels can boost cytokine production by both mouse and human lymphoid cells. Introduction Various cell media have been developed for in vitro cell cultures to optimize the growth and survival of particular cell types. For example, the RPMI1640 media is frequently used for in PLOS ONE | https://doi.org/10.1371/journal.pone.0282037 February 24, 2023 1 / 17 PLOS ONE Funding: This work was funded by grants from the Scientific and Technological Research Council of Turkey (TUBITAK, #117Z216, GW), the European Molecular Biology Organization (EMBO, #IG3073; GW), and the H2020 Marie Sklodowska-Curie Actions (#777995, GW, DS). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: αGalCer, α-galactosylceramide; FCS, fetal calf serum; ICCS, intracellular cytokine staining; iNKT, invariant Natural Killer T; MAIT, mucosal-associated invariant T; MFI, mean fluorescent intensity; PBMCs, peripheral blood mononucleated cells; ROS, reactive oxygen species; RT, room temperature. Impact of the Ca2+ concentration in vitro on lymphoid and myeloid cells vitro cultures of mouse and human lymphocytes [1–3]. However, it was suggested that the Ca2 + concentration of RPMI1640 (0.49 mM) is actually suboptimal for the in vitro stimulation of conventional mouse [4] and human [5] αβ T cells, as measured by cytokine production, and that a 1 mM CaCl2 supplement is required to obtain the maximal cytokine response. Whether the function of unconventional T cells or of other lymphoid and myeloid cells similarly is impacted by the Ca2+ concentration in vitro is currently unknown. Unconventional T cells differ from conventional αβ T cells by their development and functional capabilities. Prominent examples of unconventional T cells are invariant Natural Killer T (iNKT) cells and mucosalassociated invariant T (MAIT) cells, which both express an αβTCR, and γδ T cells, which express a γδTCR. Both iNKT and MAIT cells express a highly conserved invariant TCR αchain, which recognizes glycolipids or riboflavin derivates in the context of the non-polymorphic MHC class I homologs CD1d or MR1, respectively [6–9]. γδ T cells are largely MHCunrestricted and although the antigen for many γδ T cells is not known, some respond to phosphorylated isoprenoid metabolites or lipids [10, 11]. These unconventional T cells develop as memory T cells and can provide a first line of defence during immune responses [12]. B cells are the second main adaptive lymphoid cell type and are characterized by the expression of a BCR [13]. As an example of myeloid cells, we choose here macrophages, which are phagocytic and antigen-presenting effector cells of the innate immune system [14]. Depending on the way of stimulation, macrophages can differentiate into several functionally distinct subsets, often referred to as classically activated M1 or alternatively activated M2 macrophages [14–16]. To determine the impact of the Ca2+ concentration on lymphoid and myeloid cells besides conventional αβ T cells, we here compared their immune response in vitro in the presence of normal RPMI1640 medium (RPMInorm) and RPMI1640 medium supplemented with 1 mM Ca2+ (RPMIsuppl). Our data indicated that (...truncated)