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. 2015 Sep;146(1):173-83.
doi: 10.1111/imm.12494. Epub 2015 Jul 8.

Adenanthin, a new inhibitor of thiol-dependent antioxidant enzymes, impairs the effector functions of human natural killer cells

Marta Siernicka  1   2 , Magdalena Winiarska  1 , Malgorzata Bajor  1 , Malgorzata Firczuk  1 , Angelika Muchowicz  1 , Malgorzata Bobrowicz  1   2 , Cyril Fauriat  3 , Jakub Golab  1 , Daniel Olive  3 , Radoslaw Zagozdzon  1   4
Affiliations

Adenanthin, a new inhibitor of thiol-dependent antioxidant enzymes, impairs the effector functions of human natural killer cells

Marta Siernicka et al. Immunology. 2015 Sep.

Abstract

Natural killer (NK) cells are considered critical components of the innate and adaptive immune responses. Deficiencies in NK cell activity are common, such as those that occur in cancer patients, and they can be responsible for dysfunctional immune surveillance. Persistent oxidative stress is intrinsic to many malignant tumours, and numerous studies have focused on the effects of reactive oxygen species on the anti-tumour activity of NK cells. Indeed, investigations in animal models have suggested that one of the most important thiol-dependent antioxidant enzymes, peroxiredoxin 1 (PRDX1), is essential for NK cell function. In this work, our analysis of the transcriptomic expression pattern of antioxidant enzymes in human NK cells has identified PRDX1 as the most prominently induced transcript out of the 18 transcripts evaluated in activated NK cells. The change in PRDX1 expression was followed by increased expression of two other enzymes from the PRDX-related antioxidant chain: thioredoxin and thioredoxin reductase. To study the role of thiol-dependent antioxidants in more detail, we applied a novel compound, adenanthin, to induce an abrupt dysfunction of the PRDX-related antioxidant chain in NK cells. In human primary NK cells, we observed profound alterations in spontaneous and antibody-dependent NK cell cytotoxicity against cancer cells, impaired degranulation, and a decreased expression of activation markers under these conditions. Collectively, our study pinpoints the unique role for the antioxidant activity of the PRDX-related enzymatic chain in human NK cell functions. Further understanding this phenomenon will prospectively lead to fine-tuning of the novel NK-targeted therapeutic approaches to human disease.

Keywords: degranulation; natural killer cells; oxidative stress; peroxiredoxin; tumour surveillance.

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Figures

Figure 1
Figure 1
Peroxiredoxin 1 (PRDX1)-encoding transcript is markedly up-regulated in activated natural killer (NK) cells. Reanalysis of changes in antioxidant gene expression pattern in transcriptomic profiling in the pooled purified peripheral blood-derived CD56dim CD16+ NK, CD56bright CD16− NK and in-vitro activated (interleukin-2 + phytohaemagglutinin) CD56+ CD16+ NK subsets obtained from nine healthy donors (GEO accession number: GSE1511). The expression level for each gene in CD56dimCD16+ subset was set as 1, and the levels in the remaining two subsets are presented as the relative fold change. PRDX1, thioredoxin (TXN) and thioredoxin reductase (TXNRD1) relative expression bars in the activated NK cells are indicated with arrows. Data are presented as the averages ± SD for two technical replicates.
Figure 2
Figure 2
Adenanthin (ADNT) affects the redox balance in natural killer (NK) cells. (a) Relative reactive oxygen species (ROS) levels in NK cells incubated with hydrogen peroxide or ADNT measured by CM-H2-DCFDA fluorescence. (b) Dose- and time-dependent effects of ADNT on peroxiredoxin 1 (PRDX1) dimer/monomer presence in human NK cells, as assessed by electrophoresis in denaturing, non-reducing conditions, followed by immunoblotting. (c) Effects of N-ethylmaleimide (NEM) or ADNT on levels of glutathione (left-hand panel), glutathione disulphide (GSSG) (right-hand panel) in human NK cells. Data presented as the averages from two independent donors ± SEM. **P < 0·01.
Figure 3
Figure 3
Adenanthin (ADNT) affects natural killer (NK) cell cytotoxicity and degranulation in the pre-incubation model. To study cytotoxicity and degranulation, NK cells (isolated from three healthy donors) were stimulated overnight with cytokines, then pretreated with ADNT for 4 hr, washed three times and used as effector cells in the assays. (a) To determine the influence of ADNT on natural cytotoxicity of NK cells, CFSE-labelled K562 target cells were incubated with NK effector cells (pre-treated with ADNT) for 4 hr at an effector : target ratio of 6 : 1. Upon incubation, cells were stained with propidium iodide (PI) and analysed using flow cytometry. NK cell cytotoxicity was determined as the percentage of CFSE and PI double-positive target cells. Data are presented as the averages ± SD from three donors (left-hand graph). For the degranulation assay (right-hand graph), unstained targets were incubated for 4 hr with NK cells (pretreated with ADNT), anti-CD107a antibody and GolgiStop (at E : T ratio 1 : 1). Cells were washed, stained with anti-CD56, anti-CD3 antibodies and Flexible Viability Dye and analysed by flow cytometry. For every donor, CD107a expression in ADNT-treated groups was calculated as a percentage of CD107a-positive NK cells in the control sample of every donor. The results represent the averages of normalized results ± SD. (b) In experiments determining the influence of ADNT on NK cell cytotoxicity and degranulation in antibody-dependent cell-mediated cytotoxicity (ADCC), NK cells were incubated with Raji cells and rituximab (100 μg/ml) as described in (a). Statistical analysis was performed using the paired Student’s t-test. *P < 0·05; **P < 0·01.
Figure 4
Figure 4
Adenanthin (ADNT) decreases natural killer (NK) cell degranulation, annexin-V binding and cytokine secretion in the co-incubation model. (a) NK cell degranulation is inhibited in the presence of ADNT in natural cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. NK cells stimulated with cytokines were seeded with target cells and ADNT and incubated for 4 hr in the presence of anti-CD107a antibody and GolgiStop (at an effector : target ratio of 1 : 1). Upon incubation, cells were washed and stained as described previously. The results represent the averages of normalized results ± SD. (b) Effect of ADNT on NK cell degranulation is reversed by N-acetylcysteine (NAC) supplementation. Freshly isolated NK cells were stimulated overnight with cytokines. The next day, cells were pre-treated with NAC for 4 hr, washed and used for the degranulation assay in the presence of ADNT (4 μm) and NAC. The results represent averages of normalized results from two donors ± SD (c) In the annexin-V binding assay, NK cells isolated from the peripheral blood mononuclear cells of seven different donors were incubated alone (con) or in the presence of K562 cells for 4 hr and subsequently stained with anti-CD56 antibody and annexin-V (left graph). At right, ADNT modulates annexin-V binding to NK cells stimulated with K562 targets. NK cells were incubated alone or with K562 cells in the presence of ADNT (0, 1, 2, 4 μm) for 4 hr. Then, cells were washed and stained as described above with anti-CD56 antibody and annexin-V. The results are shown for one representative donor. Bars represent percentage of annexin-V-positive NK cells ± SD for two technical replicates. (d) Target K562 (natural cytotoxicity) or Raji cells with rituximab (100 μg/ml) (ADCC), were seeded with stimulated NK cells and ADNT for 4 hr as described in the Materials and methods section. Upon incubation, cells stained with anti-CD56, anti-CD3 and Flexible Viability Dye, were subsequently fixed, permeabilized and stained with anti-tumour necrosis factor-α (TNF-α) and anti-interferon-γ (IFN-γ) antibodies. Data are presented as a percentage of TNF-α or IFN-γ positive cells within the whole NK cell population. The experiment was performed using NK cells isolated from three different donors. Statistical analysis was performed using the paired Student’s t-test. *P < 0·05; **P < 0·01.
Figure 5
Figure 5
Adenanthin (ADNT) down-regulates expression of natural killer (NK) cell surface molecules. NK cells isolated from three healthy donors, stimulated and incubated with ADNT for 24 hr were subsequently stained with fluorochrome-conjugated antibodies and analysed using flow cytometry. The expression of each antigen was calculated as a ratio of mean fluorescence intensity (MFI) to isotype control MFI. Data are presented as the percentage of control cells. Bars indicate the averages of normalized results ± SD. Statistical analysis was performed using a paired Student’s t-test. *P < 0·05; **P < 0·01.
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