Swift Intrahepatic Accumulation of Granulocytic Myeloid-Derived Suppressor Cells in a Humanized Mouse Model of Toxic Shock Syndrome

Abstract

Toxic shock syndrome (TSS) and other superantigen-mediated illnesses are associated with ‘systemic’ immunosuppression that jeopardizes the host's ability to fight pathogens. Here, we define a novel mechanism of ‘local’ immunosuppression that may benefit the host. Systemic exposure to staphylococcal enterotoxin B (SEB) rapidly and selectively recruited CD11b⁺Gr-1^highLy-6C⁺ granulocytic myeloid-derived suppressor cells (MDSCs) to the liver of HLA-DR4 transgenic mice. Hepatic MDSCs inhibited SEB-triggered T cell proliferation in a reactive oxygen species–dependent manner, and ex vivo–generated human MDSCs also similarly attenuated the proliferative response of autologous T cells to SEB. We propose a role for MDSCs in mitigating excessive tissue injury during TSS.

Superantigens (SAgs) are exotoxins secreted by select bacterial pathogens, including Staphylococcus aureus and Streptococcus pyogenes. SAgs cause various illnesses, ranging from typically self-limiting food poisoning to potentially life-threatening TSS [1]. Moreover, if fallen into the wrong hands, SAgs can be weaponized and used against human populations. In fact, staphylococcal enterotoxin B (SEB), a prototype bacterial SAg and a cause of nonmenstrual TSS, is considered a category B priority bioterrorism agent [2].

SAgs activate a sizeable fraction of the T cell repertoire, up to 20% of exposed T cells, irrespective of their T cell receptor (TCR) specificity for complexes of cognate peptide–major histocompatibility complex (MHC). This occurs through unconventional contacts SAgs establish, in unprocessed form, with lateral surfaces of MHC class II molecules on antigen-presenting cells and select TCR β-chain variable (Vβ) domains expressed by many T cells [3, 4]. The consequent polyclonal T cell activation can lead to a cytokine storm, systemic toxicity and hyperinflammation, which is often accompanied or followed by a state of profound immunosuppression. T cells targeted by SAgs in mouse models undergo activation-induced death or become anergic [4, 5]. This generates physical or functional holes in the T cell repertoire, thus potentially compromising the host's ability to combat the very bacteria that produce SAgs, as well as concomitant or secondary infections. Regulatory/suppressor cells have also been implicated in SAg-mediated immunosuppression. For instance, in vitro exposure to SAgs reportedly converts conventional CD25⁻ T cells to interleukin-10–producing CD25⁺FoxP3⁺ regulatory T cells [6]. However, the in vivo significance of SAg-induced regulatory T cells is unclear. The potential contribution of other suppressor cell types, including myeloid-derived suppressor cells (MDSCs), to TSS-associated immunosuppression is ill-defined. Equally important, whether suppressor cell functions may benefit the host by mitigating tissue damage inflicted by unleashed, massive T cell responses remains a matter of debate. We have serendipitously discovered a dramatic, early onset and tissue-selective influx of MDSCs into the liver of HLA-DR4 transgenic (DR4 tg) mice shortly after systemic exposure to SEB. Here, we describe phenotypic and immunosuppressive characteristics of liver-infiltrating MDSCs in a humanized mouse model of TSS. We also extend our findings to an in vitro culture system in which human MDSCs modulate the T cell response to SEB.

MATERIALS AND METHODS

Ethics Statement

Peripheral blood specimens were collected from consenting healthy volunteers following a protocol approved by the Western University Research Ethics Board for Health Sciences Research Involving Human Subjects. Animal experiments were conducted in accordance with Canadian Council on Animal Care guidelines.

Animals

DR4 tg mice on a C57BL/6 (B6) background were bred in our barrier facility. These mice lack endogenous MHC class II molecules but express a chimeric HLA encoded by HLA-DRA-IEα and HLA-DRB1*0401-IEβ transgenes [7]. Adult wild-type (WT) B6 mice were purchased from Charles River and were housed and cared for in the same facility.

Mouse Model of TSS

Using an approved biosafety protocol and following the Public Health Agency of Canada regulations, recombinant SEB was cloned from S. aureus COL, expressed in Escherichia coli BL21 (DE3), and purified by nickel column chromatography [8]. As a negative control, we generated and used an inactive form of SEB that carries an N→A point mutation at position 23 [9]. This mutant, hereafter referred to as SEB_N23A, is impaired in binding to mouse TCR Vβ8.2, which is a known target of intact SEB.

Mouse MHC II molecules exhibit poor affinity for SEB [10]. Therefore, to simulate staphylococcal TSS, we injected DR4 tg mice intraperitoneally with 50 µg of SEB [8, 9]. This model may not mimic all clinical features of TSS (eg, capillary leakage due to SAgs' toxic effect on human endothelial cells), but it provides a powerful system in which to study many inflammatory and immunological aspects of TSS. Control DR4 tg animals received phosphate-buffered saline (PBS) or 50 µg of SEB_N23A. As an additional control, SEB was administered intraperitoneally to WT B6 mice.

Cytofluorimetric Analyses

Mice were euthanized by cervical dislocation. Splenic, lymph node and thymic single-cell suspensions were prepared in cold, sterile PBS. Bone marrow cells were flushed, under aseptic conditions, out of femurs and tibias. To obtain nonparenchymal hepatic mononuclear cells, livers were perfused through the central or portal vein with PBS. The tissue was then pressed through a wire mesh, and the resultant homogenate was resuspended in 33.75% Percoll Plus (GE Healthcare) and spun at room temperature. Erythrocytes among pelleted cells were lysed, and a nylon mesh strainer was used to remove clumps and debris. Cells were incubated on ice with 5 µg/mL anti-mouse CD16/CD32 monoclonal antibody (mAb; clone 2.4G2) to prevent Fcγ receptor-mediated false-positive staining with fluorochrome-labeled mAbs that were subsequently added. These included mAbs to cell surface CD11b, CD11c, CD31, CD244, CCR2, CCR5, Gr-1, Ly-6C, Ly-6G, F4/80, and c-kit. mAbs used in this study are listed in Supplementary Table 1. For detection of intracellular Ki-67 after surface staining for indicated markers, cells were fixed and permeabilized using a BD Cytofix/Cytoperm kit followed by incubation with an anti-Ki-67 mAb. The fluorescence-minus-one control was used to precisely set the gate for Ki-67⁺ events. A FACSCanto II cytometer and FlowJo software were used for data collection and analysis, respectively.

In Vitro Generation of Mouse Bone Marrow–Derived Dendritic Cells (BMDCs)

Bone marrow cells were obtained from femurs and tibias of DR4 tg mice, depleted of erythrocytes, filtered, and seeded at a density of 1 × 10⁶ cells/mL in Roswell Park Memorial Institute 1640 (RPMI 1640) medium containing 10% heat-inactivated fetal bovine serum, GlutaMax, nonessential amino acids, sodium pyruvate, penicillin and streptomycin (hereafter, “complete medium”). Cells were cultured for 6 days in complete medium containing 10 ng/mL recombinant mouse granulocyte-macrophage colony-stimulating factor and interleukin-4 (PeproTech). Every other day, nonadherent cells were discarded, and cultures were replenished with fresh medium and cytokines. On day 6, CD11c⁺ cells were magnetically purified using an EasySep Mouse CD11c Positive Selection Kit (Stemcell Technologies).

In Vitro Generation of Human MDSCs

We used the method of Lechner et al [11]. Briefly, peripheral blood mononuclear cells were isolated from heparinized whole blood by density gradient centrifugation in Ficoll-Paque Plus medium (GE Healthcare) and cultured for 7 days at 5 × 10⁵ cells per mL of complete medium supplemented with 10 ng/mL recombinant human interleukin-6 and granulocyte-macrophage colony-stimulating factor (PeproTech). Adherent cells were then gently harvested using Detachin (Genlantis) and stained with an anti-human CD33 mAb before a FACSAria III cytometer was used to sort CD33⁺ cells.

T Cell Suppression Assays

Hepatic CD11b⁺Gr-1^high cells from SEB-treated DR4 tg mice were sorted. A small aliquot was stained with Wright-Giemsa for morphological assessments. Splenic T cells from WT B6 mice were passed through nylon wool and magnetically purified using CD90.2 MicroBeads (Miltenyi Biotec). T cells were coincubated with γ-irradiated BMDCs at a 4:1 ratio of T cells to BMDCs and stimulated with 100 ng/mL SEB in complete medium for 3 days. CD11b⁺Gr-1^high cells were absent or present at indicated ratios. Cells were pulsed with tritiated thymidine ([³H]TdR) during the final 18 hours, and [³H]TdR uptake was determined by liquid scintillation counting. T cell proliferation was also judged by carboxyfluorescein succinimidyl ester (CFSE) dye dilution within the CD3ε⁺ population. In several experiments, N^G-monomethyl-L-arginine (Calbiochem), N^ω-hydroxy-nor-arginine (Calbiochem), or catalase (Sigma-Aldrich) were added to the cultures to inhibit nitric oxide synthase, arginase, or reactive oxygen species, respectively [12].

For human T cell suppression assays, peripheral blood mononuclear cells were subjected to 2 successive rounds of purification using nylon wool columns (Polysciences). T cells were seeded at 8 × 10⁴ cells/well in a 96-well plate along with 2 × 10⁴ γ-irradiated, nylon wool–adherent accessory cells, which consist mainly of B cells and monocytes. Autologous CD33⁺ MDSCs were added to the cultures before SEB treatment, and [³H]TdR uptake was quantified.

Statistical Analysis

Statistical comparisons were performed using the Student t test, and differences with a P value of <.05 were deemed statistically significant.

RESULTS AND DISCUSSION

While investigating TSS in the DR4 tg mouse model [8, 9], we found a rapid and sizable rise in a high forward- and side-scatter population among nonparenchymal hepatic mononuclear cells (Figure 1A) but not within the spleen, thymus, or lymph nodes (Supplementary Figure 1A). Two hours after SEB injection, there was a >3-fold increase in this population compared with untreated or PBS-treated DR4 tg mice (Figure 1A). This phenotype was much less pronounced in SEB-injected WT mice, indicating a requirement for higher-affinity human MHC interactions with SEB, which is in agreement with our previous reports [8, 9, 13].

Figure 1.

CD11b⁺Gr-1^highLy-6C⁺ cells amass in the liver of HLA-DR4 transgenic (DR4 tg) mice shortly after staphylococcal enterotoxin B (SEB) injection. A, Wild-type (WT) or DR4 tg mice were injected with SEB, SEB_N23A, or phosphate-buffered saline (PBS), and the frequencies of high forward-scatter (FSC) and side-scatter (SSC) cells among nonparenchymal hepatic mononuclear cells was determined 2 hours after SEB exposure. Representative dot plots are illustrated, and mean values ± standard errors of the mean for the indicated number of mice per group are shown. B, The gated population from SEB-injected DR4 tg mice was characterized cytofluorimetrically using monoclonal antibodies specific for indicated markers (open histograms) and isotype controls (filled histograms). C, The frequencies of CD11b⁺Gr-1^highLy-6C⁺ and CD11b⁺Gr-1^low/negLy-6C⁺ cells among hepatic mononuclear cells of PBS- and SEB-injected DR4 tg mice were determined. *P < .05 and ***P < .001. Abbreviation: NS, nonsignificant.

To definitively demonstrate that T cell activation by SEB is a prerequisite for this phenotype, we used SEB_N23A, a mutated version of SEB with a partially defective TCR binding capacity [9]. As anticipated, injection of DR4 tg mice with SEB_N23A failed to induce a robust enlargement in the high forward- and side-scatter population within the liver (Figure 1A). SEB_N23A retains the ability to bind to DR4, and the modest response it elicits is likely contributed by TCR Vβs other than Vβ8.2.

Next, we used a wide panel of mAbs specific for various surface markers, including but not limited to those listed in Figure 1B, to characterize the population under our scrutiny. This revealed a CD11b⁺Gr-1^highLy-6C⁺ phenotype for the vast majority of the cells, which fits the granulocytic MDSC (G-MDSC) definition [14]. The anti-Gr-1 mAb we used (clone RB6-8C5) may bind to both Ly-6G and Ly-6C. Therefore, in separate experiments, we used a different mAb (clone 1A8) and confirmed that CD11b⁺Gr-1^high cells were indeed Ly-6G⁺ and Ly-6C⁺ (Supplementary Figure 2). Furthermore, sorted cells stained with Wright-Giemsa mostly exhibited a ring-shaped nucleus (Supplementary Figure 3), which is again consistent with the G-MDSC morphology in mice [14]. It is noteworthy that a minority fraction of the high forward- and side-scatter cells were CD11b⁺Gr-1^low/negLy-6C⁺, stained positive for F4/80, and had a monocytic morphology. However, their frequencies in SEB- and PBS-treated DR4 tg mice were similar (Figure 1C).

Some of the markers expressed by MDSCs can be found on other cell types (eg, neutrophils). It was therefore crucial to verify the immunosuppressive nature of CD11b⁺Gr-1^high cells before we could define them as MDSCs. To this end, we demonstrated that CD11b⁺Gr-1^high cells sorted from the livers of SEB-treated DR4 tg mice significantly attenuated ex vivo T cell proliferation in response to SEB (Figure 2A) or a mitogenic anti-CD3ε mAb (data not shown). Using several pharmacological inhibitors, we were also able to show, mechanistically, that the suppressive effect of G-MDSCs was mediated by reactive oxygen species but not by arginase or nitric oxide synthase (Figure 2B).

Figure 2.

Mouse and human myeloid-derived suppressor cells (MDSCs) suppress staphylococcal enterotoxin B (SEB)–induced T cell proliferation. A, CD11b⁺Gr-1^high cells were sorted from the livers of HLA-DR4 transgenic (DR4 tg) mice 2 hours after injection with SEB. They were then cocultured with naive mouse T cells and in vitro-generated DR4 tg BMDCs in the presence or absence of SEB for 3 days. T cell proliferation was assayed by [³H] TdR incorporation or by carboxyfluorescein succinimidyl ester (CFSE) dye dilution. Representative data from 2 independent experiments are illustrated. Error bars correspond to standard deviations (SDs). **P < .01. B, In separate experiments, N^G-monomethyl-L-arginine (L-NMMA; 0.5 mM), N^ω-hydroxy-nor-arginine (Nor-NOHA; 0.5 mM), or catalase (1000 U/mL) were added to the cultures, and T cell proliferation was measured by [³H] TdR incorporation. Three independent experiments yielded similar results, which were pooled. Error bars represent standard errors of the mean. *P < .05. C, Human MDSCs were generated in a 7-day culture system in the presence of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) (10 ng/mL each). This method led to a dramatic expansion of CD33⁺ cells among peripheral blood mononuclear cells (PBMCs) isolated from all 3 donors recruited to this study. D, Sorted CD33⁺ cells were coincubated for 3 days with autologous T cells and accessory cells and tested for their ability to suppress SEB-triggered T cell proliferation, as described in “Materials and Methods” section. Similar results were obtained in 3 independent experiments, each of which used PBMCs from one of the 3 donors recruited to this study. Error bars represent SDs. and **P < .01 and ***P < .001. Abbreviations: FSC, forward scatter; NS, nonsignificant; SSC, side scatter.

MDSCs are known to amass in certain inflamed or tumor-bearing tissues, albeit at much later time points. The rapidity with which G-MDSCs accumulate in the liver of TSS-afflicted mice suggests their recruitment to the liver as opposed to their in situ proliferation. This notion is supported by 2 lines of evidence. First, the expression of the proliferation marker Ki-67 by CD11b⁺Gr-1^high cells was lower, rather than higher, in SEB-treated mice in comparison with controls (Supplementary Figure 4A). Second, CD11b⁺Gr-1^high cell accumulation in the liver was accompanied by a simultaneous decrease in their frequency in the bone marrow (Supplementary Figure 4B), suggesting a trafficking route from the bone marrow to the liver. The exact cellular and/or soluble factors (eg, chemokines) that mediate CD11b⁺Gr-1^high cells' recruitment to the liver during TSS remain unknown and are a subject of our ongoing investigation. Invariant natural killer T cells, which we demonstrated are directly responsive to SEB [9], compose up to 40% of the lymphocyte population in the mouse liver. However, genetic deficiency or antibody-mediated depletion of invariant natural killer T cells did not affect the G-MDSC buildup in the liver, nor did systemic depletion of CD4⁺ or CD8⁺ cells (unpublished data). Regardless, once present in the liver, G-MDSCs appear to be retained in this organ, as evidenced by their elevated frequencies even 24 hours after SEB exposure (data not shown).

The liver is uniquely tolerogenic among immunological sites. This is dictated in part by antiinflammatory properties of liver-resident cells and by expression of coinhibitory receptors that dampen T cell responses to self and foreign antigens [15]. We have now defined a novel mode of immunosuppression during TSS, which may benefit the host by diminishing hepatic tissue damage. A substantial proportion of SAg-mediated illnesses are nonmenstrual. Staphylococcal enterotoxins released in the gastrointestinal tract, due for instance to food poisoning, may cross the perturbed epithelium and access the liver through the portal vein. Therefore, it is fitting that MDSCs can swiftly and selectively home to the liver (Supplementary Figure 1A and 1B) to help protect this vital organ from SAgs' deleterious effects with inflammatory and metabolic repercussions.

Finally, ex vivo–generated human MDSCs were able to inhibit autologous T cell responses to SEB (Figure 2C and 2D), which recapitulates our findings on immunosuppressive function of sorted mouse MDSCs. Whether human liver attracts MDSCs in the early phase of TSS and/or other SAg-mediated illnesses warrants further investigation.

Supplementary Data

Supplementary materials are available at http://jid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author.