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In order to analyze S. aureus-mediated induction or inhibition of MDSC formation, we used a variety of staphylococcal strains (Table 1). Bacteria were stored as glycerol stocks at −80°C and grown overnight on TSB agar plates at 37°C (casein peptone 17 g/l, soya peptone 3 g/l, glucose 2.5 g/l, dipotassium hydrogen phosphate 2.5 g/l, sodium chloride 5 g/l, Sigma-Aldrich). Single colonies from each strain were inoculated and shaken for 16 h at 130 rpm at 37°C in RPMI 1640 medium (Biochrom) supplemented with 4 mM L-glutamine (Gibco/Life Technologies). Bacterial cells were removed by centrifugation for 30 min at 5,000x g at 4°C and the supernatants were sterile-filtered twice using 0.2 μm non-pyrogenic filters. Equivalent growth of the bacteria was verified by optical density measurements at 600 nm and by CFU counting on TSB agar plates. P. aeruginosa was grown overnight in TSB medium instead of RPMI 1640, and supernatants were prepared as described (Rieber et al., 2013). The filtered supernatants were stored in aliquots at −20°C and were used for stimulation experiments.

Where indicated, staphylococcal supernatants from RPMI overnight cultures were diluted 1:3 in RPMI 1640 and pre-treated with 20 μg/ml proteinase K (Promega) at 130 rpm at 37°C for 16 h. Heat-treatment of staphylococcal supernatants or staphylococcal enterotoxins was performed at 80°C for 20 min.

For size exclusion experiments, Vivaspin 15 concentrators with molecular weight cutoffs (MWCO) of 10,000 or 50,000 were used (Sartorius Stedim Biotech). Staphylococcal supernatants were centrifuged through MWCO 50,000 filters for 30 min at 4.000x g and 4°C, resulting in a 300-fold concentration. The flow-through was then centrifuged in a second step through MWCO 10,000 filters using the same conditions. The flow-through and the residual concentrates were adjusted after each step to the initial volume with the medium used for bacterial growth.

Peripheral blood mononuclear cells (PBMC) were prepared from heparinized human blood samples from healthy volunteers by Ficoll density gradient sedimentation (Lymphocyte separation medium, Biochrom). PBMC were washed twice in RPMI 1640 medium (Biochrom), resuspended in RPMI 1640 supplemented with 10% FCS (Biochrom), 2 mM L-glutamine (Sigma-Aldrich) and 1% penicillin/streptomycin (Biochrom), referred to as “complete medium,” and used in stimulation assays. A viability of >90% was determined for all PBMC preparations by using trypan blue staining.

Immediately after isolation PBMC were seeded into 24-well flat bottom plates (Corning) at 7.5 × 10⁵ cells per well in 1.5 ml complete medium, treated with stimulants as indicated, and cultured in a humidified atmosphere at 37°C and 5% CO₂. After 4 days, medium and stimulants were refreshed. At day 6 of cultivation, all cells were collected from PBMC cultures and washed and resuspended in D-PBS (Sigma-Aldrich). Adherent cells were harvested using non-protease Detachin Cell Detachment Solution (Genlantis). SEA and SEB were purchased from Sigma-Aldrich. Non-bacterial stimulants were granulocyte-macrophage colony-stimulating factor (GM-CSF, 10 ng/ml; Genzyme), Aspergillus fumigatus lysate (10 μg/ml; Miltenyi Biotec), and IL-2 (100 U/ml; MBL).

MDSC numbers in PBMC cultures were quantified by flow cytometric analyses. PMN-MDSC were gated as SSC^highCD33⁺CD14⁻ cells. These cells were partly positive for CD16 (see Figure 1A for gating strategy). Further representative surface marker staining demonstrated strong positivity for CD11b but negative staining for CD15 (Supplemental Figure 1). Therefore, these granulocytic cells were considered as PMN-like MDSC. Total numbers of apoptotic and dead cells were determined by annexin V and propidium iodide staining. For quantification of total live cells, trypan blue staining was performed and living cells were counted in a Neubauer chamber. PE-labeled anti-CD33, APC-labeled anti-CD11b and respective isotype control antibodies were purchased from Miltenyi Biotec; PI, FITC Annexin V, FITC-labeled anti-CD14, APC-labeled anti-CD14, FITC-labeled anti-CD15, PerCP-labeled anti-CD16, APC-labeled anti-CD3, and respective isotype control antibodies were obtained from BD Pharmingen. Flow cytometry was performed using a FACSCalibur (BD), and analyses of cell percentages were performed using BD CellQuest Pro software. The percentage of PMN-MDSC in medium-only cultures was set to 1. Percentages of PMN-MDSC after stimulation are presented as x-fold compared to medium controls. Unstained controls were used to set dot plot quadrants.

MDSC were generated from myeloid cells of the PBMC fraction as described above and isolated from cell cultures by magnetic bead cell sorting for CD33 (Miltenyi Biotec). Responder-PBMC were obtained from healthy volunteers' heparinized blood and stained with CFSE (Life Technologies) according to the manufacturer's protocol. CFSE-labeled PBMC were stimulated with 100 U/ml IL-2 (R&D Systems) and 1 μg/ml OKT3 (Janssen-Cilag). Both MDSC and CFSE-labeled PBMC were added to RPMI 1640 medium supplemented with 10% human serum, 2 mM L-glutamine, 100 IU/ml penicillin and 100 mg/ml streptomycin. In a 96-well round bottom plate (Greiner Bio-One), either 10,000/30,000 MDSC or, as a control supplemented medium only, were added to 60,000 PBMC per well. Cells were incubated in a humidified atmosphere at 37°C and 5% CO₂. On day 4 cells were harvested and stained with anti-CD8a-APC, anti-CD4-PE antibodies (BioLegend), and propidium iodide (BD). PI positive cells were excluded in flow cytometry. CFSE signals of CD4⁺ and CD8⁺ PBMC were analyzed.

Statistical analysis was performed in GraphPad Prism version 6.0 using a one-sample t-test, a Mann-Whitney test or an unpaired t-test as indicated. In all tests, differences were considered significant at P < 0.05 (^*P < 0.05; ^** P < 0.01; ^*** P < 0.001; ^**** P < 0.0001).

In order to determine the impact of S. aureus on the generation of MDSC, we built on an established in vitro MDSC generation system (Lechner et al., 2010; Rieber et al., 2013) to induce PMN-MDSC from peripheral blood of healthy donors and to quantify them by flow cytometry. PMN-MDSC were initially gated as cells with high granularity (SSC^high), bearing the myeloid marker CD33, devoid of the monocytic marker CD14 (representative dot plots Figure 1A) and with the characteristic to suppress T-cell responses (representative data in Figure 1B). Additional flowcytometric analysis revealed that these suppressive myeloid cells are strongly CD11b⁺, partly CD16⁺, and CD15⁻ (Figure 1A and Supplemental Figure S1), why we would rather term them PMN-like MDSC.

We initially screened supernatants at concentrations of 3% from a broad range of S. aureus strains, including various clinical MRSA and MSSA isolates and several mutant strains affected in their expression of prominent virulence genes for their ability to modulate the generation of MDSC. In addition, we tested supernatants from several established staphylococcal laboratory strains and from another staphylococcal species, S. carnosus. All S. aureus strains tested abrogated the formation of MDSC, except for strain RN6390, which induced MDSC (Figure 1C). In contrast, the non-pathogenic food-grade bacterium S. carnosus did not modulate MDSC cell levels significantly.

Compared to the respective wild-type strain USA300, knock out of the global virulence regulator Agr, which regulates a wide variety of virulence determinants (Novick, 2003), did not affect MDSC levels. Also, transposon mutagenesis of secA, a component of the Sec pathway for protein secretion (Green and Mecsas, 2016), had no effect in the USA300 substrain JE2.

The arginine catabolic mobile element ACME, a mobile genetic element conferring survival and growth in hosts by providing polyamine resistance (Diep et al., 2008b; Joshi et al., 2011), is predominantly expressed in MRSA strains (Shore et al., 2011). Similarly, the pore-forming pantene-valentine leucocidin (PVL), which displays toxic effects on neutrophils (Genestier et al., 2005) is present in virtually all CA-MRSA strains and in some MSSA strains (Diep et al., 2008a; Wirtz et al., 2009). Both knock out of the ACME locus in USA300 substrain SF8300 and lysogenization of PVL-negative Newman with phage ΦSa2MW, which carries the PVL gene locus lukF/S-PV (Bubeck Wardenburg et al., 2007), had no effect on the generation of MDSC compared to their respective parental strains.

Strain RN6390 is a highly-mutated member of the NCTC 8325 lineage of S. aureus strains. Its parental strain NCTC 8325 (also known as RN1) is a well-established laboratory strain commonly used for genetic studies, whereby numerous derivative strains have been engineered by means of mutagenic procedures and by reconstruction of distinct mutagenized gene loci (Herbert et al., 2010; Baek et al., 2013). To test whether any of the genes mutated had an impact on the formation of MDSC, we used supernatants of parental strain NCTC 8325 and its derivative strains RN6390, 8325-4, RN4220, HG003, and SA113 for stimulation (Figure 1D). The parental NCTC 8325 strain has functional agr, sarA, and sae global virulence regulators, however, it is characterized by a deficiency in the acitivity of the important virulence regulator sigma factor SigB, which is due to a deletion in the rsbU gene, and in the regulator gene tcaR, an activator of protein A transcription, both of which have been reconstituted in strain HG003. Moreover, it is deficient in production of beta-hemolysin due to the insertion of prophage ϕ13 in the hlb gene. ϕ13, however, is coding for the IgG, C3b and fibrin degrading staphylokinase. The strains RN6390 and 8325-4 have been cured from ϕ13 and other prophages. RN4220 displays an additional deficiency in production of functional alpha- and delta-hemolysins and a partial inactivation of the global virulence regulator region agr, whereas strain SA113 is another agr mutant derived directly from NCTC 8325. In our in vitro MDSC generation system all tested members of the 8325 family induced MDSC to a similar degree (Figure 1D). Taken together, our results suggest that the above mentioned virulence factors are not necessary for modulation of MDSC levels.

The genome of the parental NCTC 8325 strain, but not that of RN6390, has been sequenced and annotated (GenBank accession no. NC_07795.1) and protein databases are available (UniProt Taxon ID 93061). We therefore performed further comparative functional, genomic, and proteomic analyses using NCTC 8325 and the closely phylogenetically related (Baba et al., 2008) and well-characterized clinical isolates USA300, Newman and COL.

By using broad concentration ranges of S. aureus supernatants, we demonstrated that both MDSC-inhibiting and MDSC-inducing activities exist within most S. aureus strains. Lower concentrations induced PMN-MDSC (peak levels at 0.01 for USA300, 0.003 for Newman, and 0.001% for COL), whereas increased concentrations led to marked suppression of PMN-MDSC when using supernatants from these strains (Figures 2A,B). In contrast, supernatants from strain NCTC 8325 showed virtually no MDSC expanding activity below 0.01% supernatant concentration, peaked PMN-MDSC levels not until 10%, and no suppression was detected even with supernatant concentrations of up to 30%.

With rising concentrations of the supernatants we observed a concomitant proliferation of the PBMC in culture. Therefore, we did not only determine the absolute numbers of PMN-like MDSC, but also quantified total PBMC numbers and discriminated live and dead cells by trypan blue exclusion and PI staining (Figure 2). During a first phase PMN-like MDSC numbers raised, while total PBMC numbers increased concurrently and only moderate cell death occurred. During a second phase PMN-like MDSC numbers declined, whereas total PBMC still accumulated substantially despite increasing cytotoxicity. During a third phase all myeloid cells were predominantly killed when toxicity exceeded a certain level. With the exception of supernatants from strain NCTC 8325, these dose-dependent phases applied to stimulations with all tested S. aureus supernatants. In order to determine if the abrogating effect on MDSC is by inhibiting differentiation into MDSC or by actually killing of MDSC, we performed Annexin/PI staining on gated MDSC. The results in Supplemental Figure 2A point to cytotoxic effects on PMN-like MDSC rather than a stop in differentiation into MDSC. Whether this killing effect is direct or indirect via other cell types cannot be clarified within the current study, because most CD33⁺ cells, if isolated, died within three days even in the medium control (Supplemental Figure 2B). The exact mechanism for the ceasing of MDSC remains a subject for future investigations.

When different S. aureus supernatants with divergent effects were mixed, inducing activities were outweighed by suppressive activities (Figure 3A). Strain NCTC 8325 significantly raised PMN-like MDSC levels only at high concentrations. This NCTC 8325-mediated MDSC induction was suppressed by inhibitory concentrations of USA300 supernatants (Figure 3B). We concluded that, in terms of modulation of PMN-like MDSC, high concentrations of NCTC 8325 supernatants correspond to lower concentrations of supernatants derived from other S. aureus strains, and that there might be an universal factor among different S. aureus strains for dose-dependent modulation of MDSC.

To better define the nature of the MDSC modulating factor, S. aureus supernatants were pre-treated under different conditions before using them for stimulation of PBMC. After digestion with proteinase K, both the MDSC inducing and the MDSC inhibiting activities were abrogated, as shown for USA300 (Figure 4A). Heat-treatment of supernatants did not affect the results (Figure 4B). Size fraction experiments using spin columns with respective MWCO cut off pores revealed that the MDSC modulating activities resided in size fractions ranging from 10 to 50 kDa. Residual stimulating activities were irregularly observed in the fraction of larger molecules, however without statistical significance (Figure 4C).

Our results indicated that the major MDSC modulating factors of S. aureus are secreted, heat-stable, proteinaceous molecules with molecular masses between 10 and 50 kDa and are produced by pathogenic rather than by apathogenic staphylococci. Furthermore, for all strains except the NCTC 8325 family, we observed a decline of PMN-like MDSC at increasing supernatant concentrations, whereas total PBMC numbers were still growing. By immunophenotyping this proliferation was attributed amongst others to a considerable growth of CD3⁺ T-cells. The observation that heat-stable, proteinaceous factors between 10 and 50 kDa in size are responsible for modulation of PMN-like MDSC levels, and that a considerable T-cell growth occurred concomitantly to the decline in PMN-like MDSC numbers, led us to investigate if superantigenic staphylococcal enterotoxins may be the key factor involved in S. aureus-mediated modulation of MDSC.

Consistent with our results obtained for complete S. aureus supernatants, staphylococcal enterotoxin A (SEA), and enterotoxin B (SEB) dose-dependently modulated PMN-like MDSC. The MDSC modulating potencies of SEA and SEB were similar (Figure 5A). Furthermore, both enterotoxins exerted profound cytotoxic and proliferative effects on PBMC, with PMN-like MDSC numbers declining and total PBMC numbers increasing at higher concentrations. The patterns of dose-dependent changes of MDSC and total PBMC numbers mediated by enterotoxins resembled those obtained from stimulations using complete S. aureus supernatants (Figures 5B,C). Low SEA concentrations (0.1 ng/ml) induced functional, T-cell suppressive PMN-MDSC, as shown by CFSE proliferation assays (Figure 5D). In addition, when simultaneously applied, MDSC inducing concentrations were outweighed by suppressive concentrations of S. aureus supernatants and staphylococcal enterotoxins, respectively (Figure 5E). The MDSC-modulating effects of enterotoxins were heat-stable, as shown for SEA (Figure 5F).

To further elucidate the role of enterotoxins in formation of PMN-like MDSC, we used transposon mutants derived from the S. aureus strain JE2, a derivative of USA300 LAC that had been established by plasmid curing. Strain JE2 and its mutants were taken from the Nebraska transposon mutant library of S. aureus strains, which encompasses around 2,000 mutants, each harboring a transposon insertion in a distinct single gene (Fey et al., 2013). The JE2 genome includes the sek and seq genes and a selX-homolog gene, encoding staphylococcal enterotoxin K (SEK), staphylococcal enterotoxin Q (SEQ), and the enterotoxin-like protein SElX, respectively. It also contains a gene encoding a protein with homology to other enterotoxins, referred to as ‘putative enterotoxin ‘type A’. When used for stimulation of PBMC, the inactivated seq gene resulted in a 100-fold lower potency of S. aureus supernatants to modulate MDSC, whereas inactivated sek led to an ~10-fold lower potency (Figure 6A). In contrast, transposon mutagenesis of genes encoding SElX or the putative “type A” enterotoxin, which is not a homolog of SEA as determined by blastp analyses, did not affect MDSC levels compared to wild-type (Figure 6B).

Our results suggest that staphylococcal enterotoxins are crucial components in S. aureus supernatants for the modulation of MDSC levels, with staphylococcal enterotoxin Q being the strongest MDSC modulating enterotoxin in USA300 supernatants.

We examined the enterotoxin contents semi-quantitatively in the supernatants of USA300, Newman, COL, and NCTC 8325 by using mass spectrometry. After trypsin digestion of the proteins, the resulting peptides were aligned with protein sequences derived from all S. aureus strains available in UniProt database. In Table 2, all proteins detected in the supernatants and identified as staphylococcal enterotoxins (SEs) or enterotoxin-like proteins (SEls) are listed. Peptides derived from eight SEs and SEls could be identified in the culture supernatants of strain COL, where SEB turned out to be the dominant enterotoxin. In Newman, peptides derived from four SEs or SEls could be detected, with SEA and SelX being the most prominent enterotoxins. USA300 was found to express only three types of SEs or SEls, respectively. SEQ appeared to be the major enterotoxin in USA300, followed by SEK, in line with the substantially reduced PMN-MDSC-inducing capacity of the seq- mutant of USA300 substrain JE2 (Figure 6A). Only low amounts of enterotoxin-derived peptides were obtained from NCTC 8325 supernatants.

Our results show that S. aureus strains with the highest functional capacity of MDSC modulation displayed the highest total enterotoxin content determined by mass spectrometry. An overall LFQ intensity of approximately 60.5 billion units was determined for enterotoxins in COL supernatants which induced maximum PMN-like MDSC levels at concentrations of 0.001% (see Figure 2), followed by Newman (~5.0 billion units; maximum induction at 0.003%), USA300 (~1.0 billion units; maximum at 0.01%), and NCTC 8325 (~0.01 billion units; 10% of supernatant necessary for maximum MDSC induction). It has to be taken into account that for mass spectrometry analysis it was necessary to switch from RPMI to TSB medium for staphylococcal cultures in order to yield high enough bacterial density for protein analyses. Therefore the enterotoxin contents are not directly correlated between these two growth conditions as the growth rate of S. aureus influences virulence factor/enterotoxin production (Derzelle et al., 2009).

MDSC are induced by various endogenous growth factors, cytokines, and microbial stimulants. GM-CSF is an important MDSC inducing growth factor, which led to an approximately 7-fold increase in PMN-MDSC numbers in our experiments. The strong MDSC-inducing capacity of GM-CSF is dose-dependently suppressed by S. aureus-derived supernatants and enterotoxins (Figures 7A,B). In addition, PMN-MDSC suppressing concentrations of S. aureus supernatants and enterotoxins also abolished the substantial MDSC-inducing capacities of IL-2 and of preparations derived from several microbes, e.g., Pseudomonas aeruginosa supernatants or Aspergillus fumigatus lysates (Figures 7C–E). Hence we propose that S. aureus enterotoxins have a dominant effect on a diverse range of MDSC-inducing factors.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JM and handling Editor declared their shared affiliation.