Invasive Surgery Impairs the Regulatory Function of Human CD56bright Natural Killer Cells in Response to Staphylococcus aureus. Suppression of Interferon-γ Synthesis

RESEARCH ARTICLE Invasive Surgery Impairs the Regulatory Function of Human CD56bright Natural Killer Cells in Response to Staphylococcus aureus. Suppression of Interferon-γ Synthesis Renate Reinhardt1, Stephanie Pohlmann2, Holger Kleinertz2, Monika Hepner-Schefczyk2, Andreas Paul1, Stefanie B. Flohé2* 1 Department of General-, Visceral- and Transplantation Surgery, University Hospital Essen, University Duisburg-Essen, Essen, Germany, 2 Surgical Research, Department of Trauma Surgery, University Hospital Essen, University Duisburg-Essen, Essen, Germany * Abstract OPEN ACCESS Citation: Reinhardt R, Pohlmann S, Kleinertz H, Hepner-Schefczyk M, Paul A, Flohé SB (2015) Invasive Surgery Impairs the Regulatory Function of Human CD56bright Natural Killer Cells in Response to Staphylococcus aureus. Suppression of Interferon-γ Synthesis. PLoS ONE 10(6): e0130155. doi:10.1371/ journal.pone.0130155 Academic Editor: Golo Ahlenstiel, University of Sydney, AUSTRALIA Received: March 17, 2015 Accepted: May 18, 2015 Published: June 19, 2015 Copyright: © 2015 Reinhardt 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. Funding: R. Reinhardt was supported by intramural funding of the Interne Forschungsförderung Essen (IFORES). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Major surgery increases the risk for infectious complications due to the development of immunosuppression. CD56bright NK cells play a key role in the defense against bacterial infections through the release of Interferon (IFN) γ upon stimulation with monocyte-derived Interleukin (IL) 12. We investigated whether invasive visceral surgery interferes with the IFN-γ synthesis of human NK cells in response to Staphylococcus aureus. In a prospective pilot study, peripheral blood mononuclear cells (PBMC) were isolated from 53 patients before and 1 to 7 d after elective visceral surgery. The release of IL-12 and IFN-γ from PBMC upon exposure to S. aureus in vitro was quantified. The expression of the IL-12 receptor β1 chain on the surface, the phosphorylation of signal transducer and activator of transcription (STAT) 4, and the synthesis of IFN-γ on/in individual CD56bright NK cells were investigated using flow cytometry. The modulatory effect of IL-12 on the S. aureus-induced IFN-γ production in CD56bright NK cells was analyzed. The IFN-γ secretion from purified CD56bright NK cells was quantified after stimulation with IL-12 and IL-18. After surgery, CD56bright NK cells among total PBMC were impaired in the release of IFN-γ for at least 5 d. Likewise, the IL-12-induced release of IFN-γ from purified CD56bright NK cells was abolished. Upon stimulation with S. aureus, PBMC secreted less IL-12 but supplementation with recombinant IL-12 did not restore the capacity of CD56bright NK cells to produce IFN-γ. CD56bright NK cells displayed reduced levels of the IL-12Rβ1 chain whereas the phosphorylation of STAT4, the key transcription factor for the Ifng gene was not diminished. In summary, after invasive visceral surgery, CD56bright NK cells are impaired in S. aureus-induced IFN-γ production and might contribute to the enhanced susceptibility to opportunistic infections. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0130155 June 19, 2015 1 / 13 Modulation of NK Cell Function after Surgical Injury Introduction After major surgery and severe injury patients often experience infectious complications due to the development of immunosuppression [1]. Monocyte deactivation that is characterized by a reduced expression of HLA-DR molecules on the surface and a decreased release of tumornecrosis factor (TNF) α and Interleukin (IL) 12 in response to microbial stimulation is considered to contribute to immunoparalysis after severe injury [2–4]. The pathomechanisms that lead to post-surgical immunosuppression are only incompletely understood and effective therapies of nosocomial infections especially caused by multi-drug resistant strains are warranted. The successful elimination of bacteria requires the activity of cells of the innate immune system such as macrophages and neutrophilic granulocytes that ingest and eradicate microorganisms and subsequently clear the infection. Upon contact with bacteria, macrophages secrete proinflammatory cytokines such as IL-12, TNF-α, and IL-6. IL-12 activates natural killer (NK) cells for the release of Interferon (IFN) γ that in turn increases the bactericidal activity of macrophages [5]. Thus, the IL-12/IFN-γ axis plays an important role in the defense against bacterial infections. NK cells belong to the innate immune system and were originally discovered as cytotoxic cells that kill virus-infected host cells or tumor cells. Diverse subsets of human NK cells with different functions can be distinguished according to their relative expression of the adhesion molecule CD56. CD56dim NK cells are highly cytotoxic but are less potent in cytokine secretion [6]. In contrast, CD56bright NK cells have been identified as important regulatory cells in bacterial infections (reviewed in [7]) by their capability to release IFN-γ as mentioned above. NK cell-derived IFN-γ secretion is triggered by IL-12 that is released from accessory cells like monocytes/macrophages or dendritic cells [8,9]. Signaling of IL-12 through the IL-12 receptor (IL-12R) on NK cells leads to the recruitment of the Janus kinase (JAK) 2 and subsequently to the phosphorylation of the transcription factor signal transducer and activator of transcription (STAT) 4 [10]. Phosphorylated STAT4 (pSTAT4) enters the nucleus and induces the transcription of the gene for IFN-γ. In addition to cytokines, direct cell-cell contact of NK cells with accessory cells through the interaction of activating receptors such as Natural killer group 2 member D (NKG2D) on NK cells with their respective ligands on accessory cells modulates the extent of NK cell-derived IFN-γ secretion [11,12]. Staphylococcus aureus is a frequent cause of infectious complications after surgery and methicilline resistant S. aureus strains represent an increasing problem in healthcare worldwide. In this prospective pilot study, we investigated the impact of major surgery on the function of human NK cells in an in vitro model of S. aureus infection. We analyzed the synthesis of IFN-γ in circulating CD56bright NK cells before and at different time points after major visceral surgery in response to S. aureus. To address the relevance of IL-12 in this infection model, the expression of the IL-12R and downstream phosphorylation of STAT4 in CD56bright N (...truncated)