CD56^bright NK cells are suppressed in <i>Staphylococcus aureus</i>-induced IFN-γ synthesis after invasive surgery.

<p><b>(A-D)</b> PBMC were isolated from “patients 1” 24 h before (-1) and 1 d (+1), 5 d (+5), and 7 d (+7) after surgery and were stained against CD3 and CD56. <b>(A)</b> Gating strategy of CD3^-CD56^{<b>bright</b>} NK cells among total PBMC. The number indicates the percentage of gated cells in the rectangle. <b>(B)</b> Cumulative data on the percentage of gated CD3^-CD56^{<b>bright</b>} NK cells among total PBMC. <b>(C, D)</b> PBMC were cultured in the presence of <i>S</i>. <i>aureus</i> and cells were stained for intracellular IFN-γ. <b>(C)</b> Representative dot plots of intracellular IFN-γ expression in gated CD3^-CD5^{<b>bright</b>} NK cells isolated before and 1 d after injury. Numbers indicate the percentage of IFN-γ-positive (IFN-γ^<b>+</b>) cells. <b>(D)</b> Cumulative data on the kinetics of IFN-γ^<b>+</b> CD3^-CD56^{<b>bright</b>} NK cells upon exposure to <i>S</i>. <i>aureus</i> before and after injuy. <b>(E)</b> CD56^{<b>bright</b>} NK cells were purified from “patients 2” (n = 11) 1 d before and on day +1 after injury and stimulated with IL-12 and IL-18. The release of IFN-γ into the supernatant was quantified. Results are expressed as scatter plots (median, interquartile range). Statistical differences were tested using the Friedman test (B, D) or the Wilcoxon signed rank test (E). *, p<0.05; **, p<0.01; ***, p<0.001 vs. d-1. iso, isotype control antibodies.</p

Visceral surgery interferes with the responsiveness to <i>Staphylococcus aureus</i>.

<p>Peripheral blood from “patients 1” was drawn 1 d before (-1) and 1 d (+1), 5 d (+5), and 7 d (+7) after surgery. PBMC were isolated and stimulated with inactivated <i>S</i>. <i>aureus</i>. The amount of IFN-γ in the supernatant was quantified. Results are expressed as scatter plot (median, interquartile range). Statistical differences were tested using the Wilcoxon signed rank test. n = 20; *, p<0.05 vs. day -1.</p

Patients’ characteristics.

<p>Quantitative variables are expressed as median (25^th-75^th interquartile range), qualitative variables are provided as number/% of total. Significant differences between both groups were tested using Mann-Whitney and Fisher’s exact test, respectively.</p><p>Patients’ characteristics.</p

CD56^bright NK cells express reduced levels of the IL-12Rβ1 chain but increased levels of phosphorylated STAT4 after injury.

<p>PBMC were isolated from “patients 2” 24 h before (d-1) and 1 d (d+1) after injury. PBMC were stained against CD3, CD56, and IL-12Rβ1. CD3^-CD56^{<b>bright</b>} NK cells were gated as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130155#pone.0130155.g002" target="_blank">Fig 2A</a>. <b>(A)</b> Representative dot plot of IL-12Rβ1 expression on gated CD56^{<b>bright</b>} NK cells. Numbers indicate the percentage of IL-12Rβ1^<b>+</b> cells. <b>(B)</b> Cumulative data on the percentage of IL-12Rβ1^<b>+</b> cells among CD56^{<b>bright</b>} NK cells before and after injury (n = 20). <b>(C, D)</b> PBMC isolated before (d-1) and 1 d (d+1) after injury were cultured in the absence (med) or presence of <i>S</i>. <i>aureus</i>. Cells were stained against CD3, CD56, and intracellular phosphorylated STAT4 (pSTAT4). <b>(C)</b> Representative histogram of pSTAT4 staining in CD56^{<b>bright</b>} NK cells analyzed before injury. <b>(D)</b> Cumulative data on the percentage of pSTAT4-expressing (pSTAT4^<b>+</b>) cells in gated CD3^-CD56^{<b>bright</b>} NK cells (n = 6). Results are expressed as scatter plot (median, interquartile range) or as box plot (median, interquartile range, range). Statistical differences were tested using the Wilcoxon signed rank test (B) or the Friedman test (D). **, p<0.01; ##, p<0.01. iso, isotype control antibodies.</p

Invasive Surgery Impairs the Regulatory Function of Human CD56^bright Natural Killer Cells in Response to <i>Staphylococcus aureus</i>. Suppression of Interferon-γ Synthesis

<div><p>Major surgery increases the risk for infectious complications due to the development of immunosuppression. CD56^bright 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 <i>Staphylococcus aureus</i>. 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 <i>S</i>. <i>aureus in vitro</i> 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 CD56^bright NK cells were investigated using flow cytometry. The modulatory effect of IL-12 on the <i>S</i>. <i>aureus</i>-induced IFN-γ production in CD56^bright NK cells was analyzed. The IFN-γ secretion from purified CD56^bright NK cells was quantified after stimulation with IL-12 and IL-18. After surgery, CD56^bright 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 CD56^bright NK cells was abolished. Upon stimulation with <i>S</i>. <i>aureus</i>, PBMC secreted less IL-12 but supplementation with recombinant IL-12 did not restore the capacity of CD56^bright NK cells to produce IFN-γ. CD56^bright NK cells displayed reduced levels of the IL-12Rβ1 chain whereas the phosphorylation of STAT4, the key transcription factor for the <i>Ifng</i> gene was not diminished. In summary, after invasive visceral surgery, CD56^bright NK cells are impaired in <i>S</i>. <i>aureus</i>-induced IFN-γ production and might contribute to the enhanced susceptibility to opportunistic infections.</p></div