Toxic shock syndrome (TSS) is caused by staphylococcal and streptococcal superantigens (SAgs) that provoke a swift hyperinflammatory response typified by a cytokine storm. The precipitous decline in the host’s clinical status and the lack of targeted therapies for TSS emphasize the need to identify key players of the storm’s initial wave. Using a humanized mouse model of TSS and human cells, we herein demonstrate that SAgs elicit in vitro and in vivo IL-17A responses within hours. SAg-triggered human IL-17A production was characterized by remarkably high mRNA stability for this cytokine. A distinct subpopulation of CD4+ effector memory T (TEM) cells that secrete IL-17A, but not IFN-γ, was responsible for early IL-17A production. We found mouse “TEM-17” cells to be enriched within the intestinal epithelium and among lamina propria lymphocytes. Furthermore, interfering with IL-17A receptor signaling in human PBMCs attenuated the expression of numerous inflammatory mediators implicated in the TSS-associated cytokine storm. IL-17A receptor blockade also abrogated the secondary effect of SAg-stimulated PBMCs on human dermal fibroblasts as judged by C/EBP δ expression. Finally, the early IL-17A response to SAgs was pathogenic because in vivo neutralization of IL-17A in humanized mice ameliorated hepatic and intestinal damage and reduced mortality. Together, our findings identify CD4+ TEM cells as a key effector of TSS and reveal a novel role for IL-17A in TSS immunopathogenesis. Our work thus elucidates a pathogenic, as opposed to protective, role for IL-17A during Gram-positive bacterial infections. Accordingly, the IL-17–IL-17R axis may provide an attractive target for the management of SAg-mediated illnesses.

This article is featured in In This Issue, p.2517

Toxic shock syndrome (TSS) is a life-threatening illness characterized by high-grade fever, diffuse erythematous rash formation, desquamation, severe hypotension, and multiorgan dysfunction (1). It is caused by systemic exposure to bacterial toxins known as superantigens (SAgs), which are secreted by Staphylococcus aureus and Streptococcus pyogenes. TSS can be of menstrual (2) or nonmenstrual (3) origin. The vast majority of menstrual TSS cases, which are linked to high-absorbency tampon usage (4), are caused by S. aureus strains expressing the powerful SAg TSS toxin-1 (TSST-1) (5). In contrast, nonmenstrual TSS can occur with virtually any S. aureus infection and is primarily associated with TSST-1 and staphylococcal enterotoxin B (SEB) (6). The expression of streptococcal pyrogenic exotoxin A (SpeA) is also strongly correlated with streptococcal TSS (7).

SAgs are a unique family of exotoxins that activate a large proportion of T cells irrespective of their TCR specificity. Cognate peptide Ags presented in the context of self-MHC by APCs typically activate one in every 10,000 T cells. In contrast, SAgs simultaneously bind MHC class II molecules on APCs outside their Ag-binding groove (8) and select TCR Vβ domains on T cells (9). By doing so, SAgs circumvent conventional modes of Ag processing and presentation to induce the activation and proliferation of up to 50% of all exposed T cells (10). The overwhelming activation of T cells by SAgs results in excessive production of inflammatory mediators, which is commonly referred to as cytokine storm. SAgs directly stimulate the secretion of IL-2, IFN-γ, and lymphotoxin-α from T cells, as well as TNF-α, IL-1β, and IL-6 from APCs (10, 11). Additionally, SAgs initiate secondary inflammatory cytokine and chemokine responses from various nonhematopoietic cell types such as epithelial cells, endothelial cells, and fibroblasts (12). The massive and uncontrolled release of these inflammatory mediators has drastic tissue damaging effects through the activation of the coagulatory cascade, vasodilation, edema, and vascular leakage (1316). SAgs also promote the production of chemokines CXCL8, CCL2, CCL3, and CCL4 (17, 18), resulting in further recruitment of leukocytes to areas of tissue injury. The net effect of the cytokine storm is a systemic inflammatory response syndrome that may culminate in fatal multiorgan failure.

IL-17A is a potent inducer of systemic inflammation, potentiating the production or activation of inflammatory cytokines (e.g., TNF-α, IL-1β, and IL-6), chemokines, matrix metalloproteases, and transcription factors in both hematopoietic and nonhematopoietic cell types (19). IL-17A also acts synergistically with other inflammatory cytokines, including TNF-α, IL-1β, and IFN-γ, to stabilize mRNA transcripts or activate promoter regions of other inflammatory mediators (20). Although IL-17A is the archetypal cytokine of the CD4+ Th17 cell lineage (19), it can also be quickly produced by innate-like T lymphocytes such as γδ T cells, invariant NKT (iNKT) cells, and mucosa-associated invariant T (MAIT) cells (21, 22). IL-17A is often associated with early and protective host responses to bacteria, and little is known about its pathogenic potentials during infection. For example, whether exposure to SAgs elicits an early IL-17A response that may contribute to the cytokine storm is essentially unexplored.

Identification of early inflammatory mediators of the cytokine storm and their cellular sources is of utmost importance due to the rapidity with which TSS may progress to life-threatening disease. The clinical status of a SAg-exposed individual can deteriorate to multiorgan failure as early as 8–12 h after the onset of the symptoms (23), and estimates of TSS mortality range from 4 to 22% (2426). Also alarmingly, there are currently no available therapeutics to attenuate the cytokine storm, which drives the immunopathology of TSS. Therefore, understanding the critical components of this process may hold the key to designing effective therapies that reduce TSS-associated morbidity and mortality.

In this investigation, using humanized mice and human PBMCs, we found and characterized a vigorous IL-17A response that was detectable within the first few hours of SAg exposure. The kinetics of this response initially suggested the involvement of innate-like T cells (21), which we were able to rule out. Instead, we identified effector memory T (TEM) cells as the major source of mouse and human IL-17A, which were surprisingly unable to coproduce IFN-γ. IL-17A receptor blockade diminished the expression of several proinflammatory mediators by SAg-stimulated human PBMCs and also prevented the secondary upregulation of the transcription factor C/EBP δ (CEBPD) in human dermal fibroblasts. These findings suggested a pathogenic role for IL-17A in TSS, which we confirmed using a humanized mouse model of the syndrome. Therefore, to our knowledge, this work defines a previously unrecognized mechanism of TSS immunopathology that is dependent upon rapid IL-17A production by a distinct subpopulation of TEM cells.

Adult 6–12 wk-old mice closely matched for age and sex were used in all experiments. Wild-type C57BL/6 mice were purchased from Charles River Canada (Saint-Constant, QC). IL-17A–GFP mice that express enhanced GFP as a marker of IL-17A expression were obtained from The Jackson Laboratory (Bar Harbor, ME) and bred in a barrier facility at Western University. HLA-DR4 transgenic (DR4tg) mice, which are devoid of endogenous MHC class II and instead express HLA-DRA-IEα and HLA-DRB1*0401-IEβ on a C57BL/6 background (27), were bred in the same facility.

To develop a SAg-sensitive IL-17A reporter mouse strain, DR4tg and IL-17A–GFP mice were crossed. The resultant HLA-DR4 transgenic IL-17A reporter (D17) mice were bred to homozygosity and confirmed to express HLA-DR transgenes and the IL-17A–GFP reporter construct but not IAβ. In brief, genomic DNA was extracted from mouse ear clips using hot sodium hydroxide and Tris. PCR-amplified products were generated using Platinum Taq DNA polymerase (Thermo Fisher Scientific, Ottawa, ON) and primer sets that are listed in Supplemental Table I. Agarose gel electrophoresis was performed to visualize PCR products.

Wild-type C57BL/6, IL-17A–GFP, DR4tg, and D17 mice were cared for in the same facility. All animal experiments were conducted in accordance with an approved institutional animal use protocol (AUP 2010-241) and in compliance with the Canadian Council on Animal Care guidelines.

To obtain donor cells, IL-17A–GFP mice were euthanized by cervical dislocation, and bone marrow was flushed out of femurs and tibias with ice-cold PBS. Erythrocytes were lysed by addition of ACK lysis buffer for 2 min, and resulting cells were filtered through a 70-μm nylon mesh strainer. Twenty million donor cells were injected i.v. into recipient DR4tg mice, which were lethally irradiated at 9 Gy using a [137Cs] gamma irradiator before adoptive transfer. Recipient mice were maintained on drinking water supplemented with 2 mg/ml neomycin sulfate (Teknova, Hollister, CA) for 2 wk after irradiation to prevent infection.

Recombinant staphylococcal and streptococcal SAgs were generated using an approved institutional biosafety protocol that follows established Public Health Agency of Canada regulations as described previously (28). Briefly, SAgs were expressed in BL21 (DE3) Escherichia coli and purified by nickel column chromatography. As an additional control, an attenuated mutant of SEB that is impaired in binding to mouse TCR Vβ8.2 (29) was generated by site-directed mutagenesis (30). The mutant SEB carries an N→A point mutation at position 23 and is referred to as SEBN23A. LPS was purchased from Sigma-Aldrich (Oakville, ON).

Animals were injected i.p. with indicated amounts of SEB or the equivalent volume of PBS as a control. They were bled via the saphenous vein, and serum samples were obtained following centrifugation of blood at 17,000 × g for 40 min and analyzed for their cytokine content using ELISA kits from eBioscience (San Diego, CA). For in vivo IL-17A neutralization, DR4tg mice were treated with 200-μg i.p. doses of an anti–IL-17A mAb (clone 17F3; BioXCell, West Lebanon, NH) or a mouse IgG1 isotype control (clone MOPC-21) 3 h before and 1 h after they were injected with 100 μg SEB. Mice were monitored every 3 h for the initial 12 h and every 8 h thereafter for up to 7 d. Weight loss was assessed at indicated time points, and mice were sacrificed when 20% weight loss was reached following institutional protocols and Canadian Council on Animal Care guidelines. For histological analyses, mice were sacrificed at 48 h post-SEB stimulation. Liver and small intestine were extracted, fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned, and stained with H&E. Sections were scored by a board-certified veterinary pathologist who was blinded to the experimental conditions.

Peripheral blood was collected in heparinized vacutainers from healthy volunteers after their written consent was secured and following a protocol approved by the Western University Research Ethics Board for Health Sciences Research Involving Human Subjects (HSREB 5545). PBMCs were isolated by density centrifugation using 50-ml SepMate tubes from Stemcell Technologies (Vancouver, BC) and low endotoxin (<0.12 endotoxin units per ml) Ficoll-Paque Plus (GE Healthcare Life Sciences) according to the manufacturer’s instructions. PBMCs were suspended in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 0.1 mM MEM nonessential amino acids, 2 mM GlutaMAX-I, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin. PBMCs were stimulated with 100 ng/ml of indicated SAgs for up to 24 h at 37°C in polystyrene U-bottom plates at a density of 2 × 106 per well. To block IL-17 receptor A (IL-17RA) prior to SEB stimulation, PBMCs were incubated with 10 μg/ml of an anti–IL-17RA mAb (clone 133617) or mouse IgG1 isotype control (clone 11711) from R&D Systems (Minneapolis, MN) for 30 min.

For cytokine assays, culture supernatants were harvested at indicated time points and analyzed by a bead-based multiplex assay (Eve Technologies, Calgary, AB). Heat maps were generated using the matrix2png utility (31). For RNA studies, PBMCs were isolated in RNAlater (Thermo Fisher Scientific) and RNA was extracted using the Purelink RNA Mini Kit (Thermo Fisher Scientific). To eliminate contaminating genomic DNA, on-column Purelink DNase (Invitrogen Canada, Burlington, ON) was used as per the manufacturer’s instructions. RNA quantity and purity was measured by a NanoDrop ND-1000 spectrophotometer. RNA was converted to cDNA via SuperScript VILO Master Mix (Invitrogen Canada). Quantitative real-time PCR (qPCR) was performed on a StepOnePlus Real-Time PCR System using TaqMan Gene Expression Assays (Supplemental Table II) and TaqMan Fast Advanced Master Mix, all from Thermo Fisher Scientific. qPCR reactions and cycling conditions were set up according to the manufacturer’s instructions. Fold changes between conditions were calculated by the ΔΔ cycle threshold (CT) method. For low-expressing gene targets that consistently amplified with CT values >37, CT values were set to 37 for analysis.

As reliable comparisons of gene expression by qPCR require normalization to stably expressed reference genes, we first tested several commonly used reference genes for changes in SEB-stimulated PBMCs when compared with unstimulated controls. The mean fold changes of TATA-binding protein and ribosomal protein L13a (RPL13A) were no more or less than 20% (data not shown). TATA-binding protein and/or RPL13A were thus used as reference genes in our qPCR assays.

To analyze RNA stability/degradation, PBMCs were stimulated with SEB for 4 h followed by treatment with 5 μg/ml transcriptional inhibitor actinomycin D (act-D). Cells were harvested at indicated time points, RNA was extracted and converted to cDNA, and qPCR for target genes was performed as described above using 18S RNA as the reference gene.

Using an approved ethics protocol (HSREB 104888), primary human fibroblasts were derived from surgically resected normal skin samples as previously described (32). Fibroblast cultures were maintained for a maximum of six passages in DMEM medium containing 10% heat-inactivated FCS, 2 mM GlutaMAX-I and 1× Antibiotic-Antimycotic (Thermo Fisher Scientific). Fibroblasts were seeded at a density of 5 × 105 per well in a tissue culture–treated polystyrene flat-bottom plate. Four million freshly isolated PBMCs were plated into a Nunc Polycarbonate Membrane Insert (pore size: 0.4 μm), which was placed inside the fibroblast culture well. Cocultures were then stimulated with 100 ng/ml SEB. Eight hours later, the insert was removed and fibroblasts were harvested and examined by qPCR for their CEBPD expression. In several experiments, anti–IL-17RA mAb or isotype control was added to cocultures at 10 μg/ml 30 min before SEB stimulation.

PBS- and SAg-primed mice were sacrificed at indicated time points to harvest spleen, pooled lymph nodes (cervical, axillary, brachial, mesenteric, inguinal, and popliteal nodes), liver, lungs, and small intestine. Spleen and lymph nodes were homogenized using a Wheaton Dounce tissue grinder. To obtain hepatic and lung nonparenchymal mononuclear cells, tissue specimens were pressed through a wire mesh, and the extracted cells were washed, resuspended in 33.75% low endotoxin Percoll Plus (GE Healthcare Life Sciences, Mississauga, ON), and centrifuged at 700 × g for 12 min at room temperature. Pelleted cells were treated with ACK lysis buffer, washed, filtered, and resuspended in cold sterile PBS.

Intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) were isolated from the small intestine as described by Sheridan and Lefrançois (33). Briefly, the small intestine was aseptically removed, flushed with HBSS containing 5% FCS and 10 mM HEPES, digested at 37°C with 1 mM dithioerythritol, and filtered through a cell strainer (pore size: 70 μm) to generate an IEL-containing homogenate. To prepare LPLs, the remaining intestinal pieces were additionally digested, first in 1.3 mM EDTA and then in a solution containing 100 U/ml collagenase, and subsequently filtered. IEL- and LPL-containing homogenates were washed, resuspended in 44% Percoll Plus (GE Healthcare Life Sciences), underlaid with 67% Percoll Plus, and spun at 600 × g for 20 min. IELs and LPLs were harvested by a Pasteur pipette from the buffy coat formed at the gradient interface, washed, and resuspended in cold PBS.

Freshly isolated mouse cells were incubated with 5 μg/ml of an anti-mouse CD16/CD32 mAb (clone 2.4G2) to block Fcγ receptors and stained at 4°C with fluorochrome-conjugated mAbs to cell surface molecules listed in Supplemental Table II. Corresponding isotype controls were used in parallel for appropriate gating. A FACSCanto II cytometer (BD Biosciences, Mississauga, ON) was used for data collection. Data were analyzed by FlowJo software (Tree Star, Ashland, OR).

Freshly isolated human PBMCs were stimulated with SAgs for 4 h, unless otherwise stated, in the presence of 1 μM brefeldin A (Sigma-Aldrich). Cells were thoroughly washed and stained at 4°C with fluorochrome-conjugated mAbs to cell surface molecules (Supplemental Table II). For intracellular detection of cytokines, the Intracellular Fixation and Permeabilization Buffer Set from eBioscience was used. We also employed a Mouse IL-17A Secretion Assay kit from Miltenyi Biotec (Auburn, CA) to confirm the secretion of this cytokine. For detection of transcription factors, the FoxP3 Staining Buffer Set (eBioscience) was used. For RNA staining, the Prime FlowRNA Assay (eBioscience) was used with probes designed for the detection of IL-17A, IFN-γ and RPL13A signals. Cells were treated and stained for surface and intracellular molecules (Supplemental Table II) following the manufacturer’s instructions. Isotype and/or fluorescence minus one controls were used in parallel for proper gating. Samples were run on a BD FACSCanto II or a BD LSR II (when using violet fluorochrome-conjugated mAbs) and analyzed by FlowJo software (Tree Star).

Analyses were carried out using GraphPad Prism 7 software (La Jolla, CA). The Shapiro–Wilk test was performed to verify the normal distribution of our data sets. Statistical comparisons were made using parametric Student t tests, ANOVA with Holm–Sidak post hoc analysis, or log-rank test, as appropriate and as indicated in the figure legends. Differences with p ≤ 0.05 were deemed statistically significant.

SAgs have notoriously poor binding affinity for MHC class II molecules expressed in certain conventional mouse strains such as C57BL/6 mice (34). Thus, we investigated the pathogenesis of TSS in humanized DR4tg mice that allow for high-affinity interactions with SAgs and consequently recapitulate many aspects of TSS-mediated immunopathology (28, 29, 3539). Using this model, we serendipitously found a dramatic serum IL-17A spike at 2 h post-SEB exposure (Fig. 1A). Given that the role of IL-17A in TSS is unknown and that IL-17A induces and synergizes with many inflammatory mediators implicated in the cytokine storm, we sought to fully characterize this phenomenon. IL-17A was present in the serum at 2 h but not detectable at 12 or 24 h after i.p. injection of SEB (Fig. 1A). In contrast, IL-17A was not detectable in SEB-injected wild-type C57BL/6 mice (Fig. 1A) and neither were several other cytokines tested (e.g., IFN-γ and IL-4). This highlights the requirement for human MHC class II molecules in the optimal activation of T cells by SAgs. The injection of SEBN23A, a mutant version of SEB with impaired binding to Vβ8.2 TCR (29), into DR4tg mice also resulted in no detectable IL-17A in the serum (Fig. 1A), indicating that SEB-induced IL-17A production is highly T cell dependent.

FIGURE 1.

Rapid IL-17A production by CD4+ TEM cells in a humanized mouse model of TSS. (A) DR4tg mice were injected i.p. with 50 μg SEB, SEBN23A, or PBS, and bled at indicated time points for serum cytokine measurements. Mean ± SEM values from two independent experiments are shown (n = 4). (BD) IL-17A–GFP/DR4tg chimeric mice were injected i.p. with PBS or 50 μg SEB and sacrificed after 2 h. (B) Serum IL-17A levels in SEB- and PBS-treated IL-17A–GFP/DR4tg bone marrow chimeric mice were determined by ELISA. Mean values ± SEM from three independent experiments are shown (n = 4–5 mice per group). (C) The frequencies of IL-17A–GFP+ cells and the geometric MFI of IL-17 were determined by flow cytometry among nonparenchymal mononuclear cells (MNCs) obtained from indicated organs of SEB- and PBS-treated mice. Mean values ± SEM from three independent experiments are shown (n = 6–7 per group). *p ≤ 0.05, two-tailed Student t test. (D) The phenotype of IL-17A–GFP+ cells among IELs from SEB-treated mice was determined using fluorochrome-conjugated Abs and tetramers for indicated markers (open black histograms) and isotype controls (filled, gray histograms). Representative dot plot and histograms from three independent experiments are shown (n = 4–6). N.D., not detectable.

FIGURE 1.

Rapid IL-17A production by CD4+ TEM cells in a humanized mouse model of TSS. (A) DR4tg mice were injected i.p. with 50 μg SEB, SEBN23A, or PBS, and bled at indicated time points for serum cytokine measurements. Mean ± SEM values from two independent experiments are shown (n = 4). (BD) IL-17A–GFP/DR4tg chimeric mice were injected i.p. with PBS or 50 μg SEB and sacrificed after 2 h. (B) Serum IL-17A levels in SEB- and PBS-treated IL-17A–GFP/DR4tg bone marrow chimeric mice were determined by ELISA. Mean values ± SEM from three independent experiments are shown (n = 4–5 mice per group). (C) The frequencies of IL-17A–GFP+ cells and the geometric MFI of IL-17 were determined by flow cytometry among nonparenchymal mononuclear cells (MNCs) obtained from indicated organs of SEB- and PBS-treated mice. Mean values ± SEM from three independent experiments are shown (n = 6–7 per group). *p ≤ 0.05, two-tailed Student t test. (D) The phenotype of IL-17A–GFP+ cells among IELs from SEB-treated mice was determined using fluorochrome-conjugated Abs and tetramers for indicated markers (open black histograms) and isotype controls (filled, gray histograms). Representative dot plot and histograms from three independent experiments are shown (n = 4–6). N.D., not detectable.

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We next tested a panel of bacterial SAgs other than SEB for their ability to induce IL-17A production in DR4tg mice. These included TSST-1, staphylococcal enterotoxin A (SEA), SpeA, streptococcal pyrogenic exotoxin I (SpeI), and streptococcal mitogenic exotoxin Z (SmeZ). TSST-1 administration did not result in IL-17A appearance in the serum and neither did SpeI or SmeZ (data not shown). As such, DR4tg mice do not appear to provide a suitable model in which to study the role of IL-17A in the context of TSST-1–mediated TSS or SpeI/SmeZ-triggered host responses. In contrast, animals injected with SEA and SpeA had detectable IL-17A levels in their serum (55 and 286 pg/ml, respectively). Therefore, the early IL-17A production may not be limited to staphylococcal TSS, and may also play a role in streptococcal infections. Of note, LPS, a potent stimulus that activates immune cells through a different mechanism than SAgs, failed to trigger an IL-17A response in DR4tg mice (data not shown).

To identify the cellular source of IL-17A, we generated IL-17A–GFP/DR4tg bone marrow chimeric mice. This system allows for quantification of IL-17A responses as measured by GFP fluorescence in SAg-sensitive, human MHC class II–expressing mice. Three weeks after the adoptive transfer of bone marrow cells, chimeric mice were injected with 50 μg SEB and sacrificed 2 h later to obtain blood, spleen, lymph nodes (cervical, axillary, brachial, mesenteric, inguinal, and popliteal), liver, lungs, and small intestine. When compared with PBS controls, which showed no detectable IL-17A in serum, SEB-injected chimeric mice produced substantial amounts of IL-17A after 2 h (Fig. 1B). The greatest frequency of IL-17A–expressing cells was found among IELs of the small intestine (Fig. 1C). Notably, the frequencies of IL-17A–expressing cells did not significantly differ between SEB and PBS treatment groups (Fig. 1C). However, the mean fluorescence intensity (MFI) of GFP, an indicator of IL-17A production on a per cell basis, increased upon in vivo SEB exposure in IELs and to a lesser degree among lung and splenic nonparenchymal mononuclear cells (Fig. 1C).

Having determined that IL-17A expression was highly upregulated and most abundant in the IEL compartment after SEB exposure, we next sought to immunophenotype these rapidly IL-17A–producing cells. The majority of IL-17A+ cells were defined as CD4+ TEM cells due to their TCRβ+CD4+CD44+CCR7CD62LCD127+ surface phenotype (Fig. 1D) (40). Similarly, CD4+ TEM cells constituted the majority of IL-17A+ cells among IELs of PBS-treated mice (data not shown).

We previously reported that iNKT cells can be activated by group II bacterial SAgs, including SEB, leading to a swift cytokine response (29). In addition, a subset of iNKT cells is capable of producing IL-17A in response to glycolipid Ags (41). Therefore, it was important to rule in or rule out the participation of iNKT-17 cells in our model. We found that in vivo stimulation with SEB gives rise to IL-17A–expressing cells that do not possess the canonical TCR of iNKT cells as judged by their lack of staining with α-galactosylceramide–loaded CD1d tetramer (Fig. 1D).

As a confirmatory approach, we generated D17 mice by crossing DR4tg mice with IL-17A–GFP reporter animals. We found a pattern similar to that observed in the bone marrow chimeras, namely increased IL-17A MFI among IELs of SEB-primed D17 mice (Supplemental Fig. 1). IL-17A–producing cells in D17 mice also similarly exhibited a TEM phenotype. Having an adequate supply of D17 animals enabled us to additionally investigate the lamina propria, a site in which IL-17A–secreting T cells are known to be abundant (42). These experiments revealed that systemic exposure to SEB raises both the frequency of IL-17A+CD4+ TEM cells in the lamina propria and the intensity of IL-17A production by these cells. In fact, the IL-17A response was stronger in the LPL compartment in comparison with IELs (Supplemental Fig. 1).

Together, the above findings show that select SAgs induce rapid IL-17A production by CD4+ TEM cells in a clinically relevant mouse model of TSS. The IL-17A response by mouse CD4+ TEM cells requires high-affinity interactions between SAgs and human MHC, and is most pronounced among intestinal IELs and LPLs.

After we established that SAgs were able to stimulate early IL-17A production in DR4tg mice, we assessed whether SAgs also elicit IL-17A responses in human PBMCs. First, PBMCs from healthy donors were stimulated with SEB or TSST-1 and analyzed for changes in cytokine mRNA levels by qPCR. Samples from all donors showed massive IL-17A mRNA upregulation after stimulation by SEB and, to a slightly lesser extent, by TSST-1 (Fig. 2A). IL-17A mRNA was highly upregulated at 2 h, reached a maximum at 4 h, and gradually decreased until 24 h poststimulation (Fig. 2A). Interestingly, the magnitude and the kinetics of the IL-17A mRNA response differed considerably when compared with IFN-γ (Fig. 2A, 2B), a prototypic proinflammatory cytokine that is induced by SAgs in humans (43) and that is considered pathogenic in mouse models of TSS (44). IL-17A mRNA upregulation was far greater in magnitude at early time points and began to decline after 4 h whereas IFN-γ mRNA levels steadily increased until 24 h (Fig. 2A, 2B).

FIGURE 2.

SAgs induce rapid IL-17A synthesis by human PBMCs. Human PBMCs were left untreated or stimulated with 100 ng/ml SEB or TSST-1 for indicated durations. (A) IL-17A or (B) IFN-γ mRNA upregulation in SAg-stimulated PBMCs was determined by qPCR. Data are shown as fold change relative to unstimulated medium control at each time point. (C and D) Cytokine concentrations in the supernatant of SAg-stimulated PBMCs at indicated time points were determined by bead-based cytokine multiplexing. Mean values ± SEM from four individual donors are shown. Statistical comparisons were made by two-way ANOVA (p ≤ 0.0001) with Holm–Sidak post hoc analysis). (E) Human PBMCs were stimulated with SEB for 4 h prior to act-D treatment. IL-17A and IFN-γ mRNA fold changes normalized to time zero (i.e., addition of act-D) were determined by qPCR. Data pooled from three individual donors are shown. Statistical comparisons were made by two-way ANOVA (p = 0.0072) with Holm–Sidak post hoc analysis. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

FIGURE 2.

SAgs induce rapid IL-17A synthesis by human PBMCs. Human PBMCs were left untreated or stimulated with 100 ng/ml SEB or TSST-1 for indicated durations. (A) IL-17A or (B) IFN-γ mRNA upregulation in SAg-stimulated PBMCs was determined by qPCR. Data are shown as fold change relative to unstimulated medium control at each time point. (C and D) Cytokine concentrations in the supernatant of SAg-stimulated PBMCs at indicated time points were determined by bead-based cytokine multiplexing. Mean values ± SEM from four individual donors are shown. Statistical comparisons were made by two-way ANOVA (p ≤ 0.0001) with Holm–Sidak post hoc analysis). (E) Human PBMCs were stimulated with SEB for 4 h prior to act-D treatment. IL-17A and IFN-γ mRNA fold changes normalized to time zero (i.e., addition of act-D) were determined by qPCR. Data pooled from three individual donors are shown. Statistical comparisons were made by two-way ANOVA (p = 0.0072) with Holm–Sidak post hoc analysis. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

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To ascertain whether human IL-17A was actively secreted among other rapidly produced inflammatory mediators (43), culture supernatants from SAg-stimulated PBMCs were assayed for cytokines by bead-based multiplexing. Both SEB and TSST-1 provoked rapid IL-17A and IFN-γ responses, which were detectable as early as 2 h and continued to accumulate by 24 h (Fig. 2C). The inflammatory cytokines TNF-α and IL-6, as well as the growth-promoting cytokine IL-2, were also produced within hours of SAg stimulation (Fig. 2D). Although the kinetics of cytokine expression was consistent for both SEB and TSST-1 stimulation, SEB elicited a more robust response for most cytokines tested when compared with TSST-1 (Fig. 2C, 2D).

Thus far, our findings revealed an unexpected discordance vis-à-vis the kinetics of IL-17A expression at mRNA and protein levels; that is, whereas IL-17A mRNA was rapidly upregulated and gradually declined after 4 h, IL-17A protein continued to accumulate in the supernatant up to 24 h (Fig. 2A, 2C). This discordance was especially apparent when compared with the IFN-γ expression kinetics that followed a similar pattern for both cytokine mRNA and protein (Fig. 2B, 2C). We hypothesized that disparities in mRNA stability between IL-17A and IFN-γ may account for the noted differences as more stable mRNA may not require consistent upregulation for translation into increasing amounts of protein. Because the degree of IL-17A mRNA stability in primary human T cells was unknown, we tested our hypothesis by comparing the half-lives of IL-17A and IFN-γ mRNA in SEB-stimulated PBMC cultures using the transcriptional inhibitor act-D (45). PBMCs were challenged with SEB for 4 h consistent with the IL-17A mRNA response peak (Fig. 2A), treated with act-D, and analyzed for cytokine mRNA expression in the subsequent 8 h after transcriptional arrest. Intriguingly, whereas IFN-γ mRNA was quickly degraded within the first few hours, IL-17A mRNA expression remained remarkably stable after the addition of act-D (Fig. 2E).

These results indicate that SAgs potently activate human PBMCs to rapidly upregulate IL-17A mRNA and secrete IL-17A protein within hours. Interestingly, the initial IL-17A mRNA upregulation is greater in magnitude and yields more stable mRNA in comparison with the canonical inflammatory cytokine IFN-γ although both cytokines are quickly secreted in culture.

As this work provides the initial report of IL-17A secretion in human PBMCs within the first few hours of SAg exposure, we next aimed to identify its cellular source. Exposure to either SEB or TSST-1 resulted in substantial intracellular IL-17A production by CD3+ T cells within 4 h (Fig. 3A). SEB was significantly more potent than TSST-1 and caused IL-17A production by 0.182% of all PBMCs (Fig. 3A). The MFI of IL-17A staining was equivalent though, indicating that SEB- and TSST-1–stimulated cells synthesized similar amounts of IL-17A on a per cell basis (Fig. 3A). We also observed that IL-17A+ cells among TSST-1–stimulated PBMCs showed reduced CD3 expression suggesting partial TCR downregulation. Lastly, we confirmed that only CD3+ T cells were actively secreting IL-17A using a cytokine secretion assay in which IL-17A that is about to be secreted binds to the cell surface via a bivalent Ab molecule specific for both CD45 and IL-17A (data not shown).

FIGURE 3.

CD4+ TEM cells are the major source of IL-17A in SAg-stimulated human PBMCs. Human PBMCs were left untreated in medium or stimulated with 100 ng/ml SEB or TSST-1 for 4 h. (A) IL-17A–producing cells were identified by intracellular cytokine staining and further analyzed by flow cytometry. Representative dot plots of SAg-stimulated PBMCs are depicted. The percentage of IL-17A–expressing cells and geometric MFI of IL-17A are shown as mean values ± SEM for six individual donors. One-way ANOVA (p = 0.0007) with Holm–Sidak post hoc analysis was used (* p ≤ 0.05, **p ≤ 0.01); n.s., not significant by two-tailed Student t test. (B) IL-17A+ cells were immunophenotyped based on their expression of indicated cell surface and intracellular markers (open red histograms) relative to isotype control (filled, gray histograms). Illustrated data represent four individual donors displaying similar results. (C) Values represent mean percentages ± SEM of four individual donors. Subtypes were defined as: CD4+ TEM (CD4+CD3+CD45RO+CCR7), CD4+ TCM (CD4+CD3+CD45RO+CCR7+), γδ T (γδ TCR+), iNKT (CD3+CD1d tetramer+), MAIT (CD3+Vα7.2 TCR+CD161+), CD8+ T (CD3+CD4CD8+). (D) IL-17A protein and mRNA coexpressing cells were identified using the PrimeFlow RNA technology and analyzed by flow cytometry. Dot plots are representative of four individual donors. TCM, central memory T cells.

FIGURE 3.

CD4+ TEM cells are the major source of IL-17A in SAg-stimulated human PBMCs. Human PBMCs were left untreated in medium or stimulated with 100 ng/ml SEB or TSST-1 for 4 h. (A) IL-17A–producing cells were identified by intracellular cytokine staining and further analyzed by flow cytometry. Representative dot plots of SAg-stimulated PBMCs are depicted. The percentage of IL-17A–expressing cells and geometric MFI of IL-17A are shown as mean values ± SEM for six individual donors. One-way ANOVA (p = 0.0007) with Holm–Sidak post hoc analysis was used (* p ≤ 0.05, **p ≤ 0.01); n.s., not significant by two-tailed Student t test. (B) IL-17A+ cells were immunophenotyped based on their expression of indicated cell surface and intracellular markers (open red histograms) relative to isotype control (filled, gray histograms). Illustrated data represent four individual donors displaying similar results. (C) Values represent mean percentages ± SEM of four individual donors. Subtypes were defined as: CD4+ TEM (CD4+CD3+CD45RO+CCR7), CD4+ TCM (CD4+CD3+CD45RO+CCR7+), γδ T (γδ TCR+), iNKT (CD3+CD1d tetramer+), MAIT (CD3+Vα7.2 TCR+CD161+), CD8+ T (CD3+CD4CD8+). (D) IL-17A protein and mRNA coexpressing cells were identified using the PrimeFlow RNA technology and analyzed by flow cytometry. Dot plots are representative of four individual donors. TCM, central memory T cells.

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To immunophenotype IL-17A–producing cells, PBMCs were challenged with SEB for 4 h and analyzed for a variety of surface markers and transcription factors by flow cytometry. The vast majority of IL-17A+ cells expressed high levels of CD4 and CD45RO, and were CCR7 (Fig. 3B), indicating a surface phenotype characteristic of CD4+ TEM cells (46). In line with previous studies (4749), IL-17A+ cells also expressed CCR6 and the transcription factor RORγT (Fig. 3B), which are hallmarks of the Th17 lineage. Notably, the majority of IL-17A+ cells expressed the lectin receptor CD161 (Fig. 3B). CD161 is highly expressed on innate-like T cells including γδ T, MAIT, and iNKT cells but only moderately expressed on Th17 cells (50). Thus, we investigated γδ TCR, Vα7.2 TCR and iNKT TCR expression to rule in/out γδ T, MAIT, and iNKT cells, respectively (Fig. 3B). These innate-like T cell subsets, in addition to a small population of CD8+ T cells, constituted only minor fractions of the overall IL-17A+ cell population (Fig. 3C). In contrast, CD4+ TEM cells were the dominant IL-17A–expressing population, which comprised >75% of IL-17A+ cells (Fig. 3C).

Strikingly, our results revealed thus far that only a small fraction of PBMCs produced IL-17A protein in response to SAgs (Fig. 3A) despite the massive upregulation of IL-17A mRNA observed in bulk PBMCs (Fig. 2A). We initially postulated that other cell types in addition to CD4+ TEM cells contributed to the substantial IL-17A mRNA response, but were not detectable by intracellular IL-17A staining. To address this, we used the PrimeFlow RNA assay, a new technology that utilizes fluorescent in situ hybridization to enable simultaneous detection of cells expressing IL-17A mRNA and intracellular IL-17A protein by flow cytometry. As expected, SEB stimulation enhanced the expression of IL-17A mRNA (Fig. 3D). Moreover, CD4+ TEM cells (CD3+CD45RO+CCR7) comprised the majority of IL-17A mRNA+ cells (Fig. 3D), consistent with our intracellular IL-17A protein findings (Fig. 3C). Notably, a small proportion of IL-17A mRNA-producing CD4+ T cells stained positively for CCR7 (Fig. 3D), indicating that CD4+ central memory T cells may be a source of IL-17A protein at later time points. These results indicate that CD4+ TEM cells are primarily responsible for rapid IL-17A secretion among SAg-stimulated PBMCs despite their relative rarity.

As multicytokine-producing T cells are reportedly induced by SEB stimulation at relatively late time points (51), we explored whether early IL-17A producers would also coexpress IFN-γ. Surprisingly, only a minute subset of IL-17A/IFN-γ coexpressors was detectable following SAg stimulation over a 4 h period, and the overwhelming majority of cytokine-producing cells exclusively expressed either IL-17A or IFN-γ (Fig. 4A). A similar trend for IL-17A and IFN-γ expression was also evident at 24 h post–SAg stimulation (Fig. 4B). Interestingly, at 4 h post-SEB, CD4+ TEM cells constituted a large proportion of IFN-γ+ cells (Fig. 4C), similar to IL-17A+ cells observed above. This finding prompted us to examine whether IL-17A–producing CD4+ TEM cells had the potential to produce IFN-γ mRNA, but were not synthesizing appreciable amounts of IFN-γ protein. We found that only a small fraction of IL-17A+ cells showed detectable IFN-γ mRNA transcripts (Fig. 4D).

FIGURE 4.

SAg-stimulated IL-17A– and IFN-γ–producing cells are two distinct populations. Human PBMCs from healthy volunteers were left untreated or stimulated with 100 ng/ml SEB or TSST-1 for 4 h (A, C, and D) or 4 h and 24 h (B). IL-17A– and IFN-γ–producing cells were identified by intracellular cytokine staining and quantified by flow cytometry. Representative dot plots of SAg-stimulated PBMCs and mean percentages ± SEM from two to four individual donors are shown. Statistical comparisons were made by one-way ANOVA (p = 0.0255 and p = 0.0224 for SEB and TSST-1 treatment conditions, respectively) with Holm–Sidak post hoc analysis (*p ≤ 0.05). (C) Gated IFN-γ–producing cells were immunophenotyped by their expression of surface markers (open black histograms) relative to isotype control (filled, gray histograms). Data are representative of four individual donors. (D) IL-17A and IFN-γ mRNA coexpressing cells were identified using the PrimeFlow RNA technology and analyzed by flow cytometry. Dot plots are representative of four individual donors.

FIGURE 4.

SAg-stimulated IL-17A– and IFN-γ–producing cells are two distinct populations. Human PBMCs from healthy volunteers were left untreated or stimulated with 100 ng/ml SEB or TSST-1 for 4 h (A, C, and D) or 4 h and 24 h (B). IL-17A– and IFN-γ–producing cells were identified by intracellular cytokine staining and quantified by flow cytometry. Representative dot plots of SAg-stimulated PBMCs and mean percentages ± SEM from two to four individual donors are shown. Statistical comparisons were made by one-way ANOVA (p = 0.0255 and p = 0.0224 for SEB and TSST-1 treatment conditions, respectively) with Holm–Sidak post hoc analysis (*p ≤ 0.05). (C) Gated IFN-γ–producing cells were immunophenotyped by their expression of surface markers (open black histograms) relative to isotype control (filled, gray histograms). Data are representative of four individual donors. (D) IL-17A and IFN-γ mRNA coexpressing cells were identified using the PrimeFlow RNA technology and analyzed by flow cytometry. Dot plots are representative of four individual donors.

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Collectively, the above results indicate that SAg-based stimulation of human PBMCs induces a rapid and potent IL-17A response that is chiefly mediated by CD4+ TEM cells. Although CD4+ TEM cells are responsible for substantial early IFN-γ production as well, the IL-17A– and IFN-γ–producing subsets were two independent populations.

Many downstream targets of IL-17A signaling are inflammatory cytokines and chemokines (52), which likely augment or perpetuate the overwhelming inflammatory cascade encountered in TSS. Accordingly, stimulation of human PBMCs with SEB for 8 h resulted in upregulation of many different pro- and anti-inflammatory genes known to be modulated by IL-17A (Supplemental Table III). Also highly upregulated was the related inflammatory cytokine IL-17F, which signals through the same IL-17RA/IL-17RC complex (53). Thus, we sought to evaluate the contribution of IL-17RA signaling to induction of inflammatory genes in the initiation of the cytokine storm.

PBMCs were preincubated with a blocking IL-17RA mAb or isotype control, stimulated with SEB for 8 h, and evaluated for changes in gene expression by qPCR. IL-17RA blockade resulted in diminished expression of several key inflammatory cytokines involved in TSS, including TNF-α, IFN-γ, and IL-6 (Fig. 5A). Additionally, the expression of many chemokines such as CCL5, CCL7, CXCL1, and CXCL6 were highly downregulated. Interestingly, the blockade of IL-17RA signaling also resulted in upregulation of notable anti-inflammatory cytokines IL-4, TGF-β1, and IL-10 (Fig. 5A). It is noteworthy that in these experiments, we chose to work with a mAb dose (i.e., 10 μg/ml) that was previously reported to efficiently block IL-17RA (54, 55). In a limited number of experiments, we reconfirmed that this dose could significantly inhibit the upregulation of CEBPD, a transcription factor linked to acute inflammatory conditions (56), in SEB-exposed PBMCs. In contrast, a 0.1 μg/ml dose of anti–IL-17RA was completely ineffective, and a 1 μg/ml dose inhibited CEBPD upregulation only marginally (data not shown).

FIGURE 5.

Blockade of IL-17RA signaling attenuates SEB-induced inflammatory gene expression in human PBMCs and in downstream nonhematopoietic cells. (A) Human PBMCs were preincubated with 10 μg/ml anti–IL-17RA blocking Ab or isotype control and subsequently stimulated with 100 ng/ml SEB for 8 h. Changes in gene expression were determined by qPCR and represented as log2 (fold change) of the anti–IL-17RA group normalized to isotype control. Differentially regulated genes were defined as a >10% fold change difference between treatment groups. Fold changes in gene expression were pooled from three individual donors and are represented as mean values. (B) Human dermal fibroblasts were grown in a flat-bottom plate and cocultured with human PBMCs that were seeded into a cell culture insert with a permeable membrane. Fibroblasts and PBMCs were in two physically separate compartments. Cocultures were left untreated or stimulated with 100 ng/ml SEB. Eight hours later, the insert was removed and fibroblasts were harvested and examined by qPCR for their CEBPD expression. Anti–IL-17RA or isotype control (10 μg/ml each) was added 30 min before SEB stimulation as indicated. Fold changes in CEBPD gene expression relative to the fibroblast-only condition was calculated for three PBMC samples prepared from three individual donors. Data are shown as mean ± SEM (* p ≤ 0.05, two-tailed Student t test).

FIGURE 5.

Blockade of IL-17RA signaling attenuates SEB-induced inflammatory gene expression in human PBMCs and in downstream nonhematopoietic cells. (A) Human PBMCs were preincubated with 10 μg/ml anti–IL-17RA blocking Ab or isotype control and subsequently stimulated with 100 ng/ml SEB for 8 h. Changes in gene expression were determined by qPCR and represented as log2 (fold change) of the anti–IL-17RA group normalized to isotype control. Differentially regulated genes were defined as a >10% fold change difference between treatment groups. Fold changes in gene expression were pooled from three individual donors and are represented as mean values. (B) Human dermal fibroblasts were grown in a flat-bottom plate and cocultured with human PBMCs that were seeded into a cell culture insert with a permeable membrane. Fibroblasts and PBMCs were in two physically separate compartments. Cocultures were left untreated or stimulated with 100 ng/ml SEB. Eight hours later, the insert was removed and fibroblasts were harvested and examined by qPCR for their CEBPD expression. Anti–IL-17RA or isotype control (10 μg/ml each) was added 30 min before SEB stimulation as indicated. Fold changes in CEBPD gene expression relative to the fibroblast-only condition was calculated for three PBMC samples prepared from three individual donors. Data are shown as mean ± SEM (* p ≤ 0.05, two-tailed Student t test).

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In the next series of experiments, we used a human PBMC-dermal fibroblast coculture system to mimic the secondary effect of IL-17A signaling in nonhematopoietic cells. To this end, PBMCs and fibroblasts were cultured in physically separate compartments prior to SEB stimulation. We found an ∼3-fold increase in the expression level of CEBPD by dermal fibroblasts that were cocultured with SEB-primed PBMCs (Fig. 5B). Importantly, this response was abrogated in the presence of 10 μg/ml anti–IL-17RA mAb. Also as expected, SEB did not change the expression of CEBPD in fibroblasts alone, a condition that was used as a control (Fig. 5B).

Taken together, the above findings strongly suggest that IL-17RA signaling plays an important proinflammatory role in TSS by increasing the expression of inflammatory mediators that are expected to contribute to TSS pathogenesis.

Because the cytokine storm drives many symptoms of TSS, attenuation of rapidly produced cytokines that further perpetuate the inflammatory cascade, such as IL-17A, may be of prime importance to reduce TSS severity. Furthermore, IL-17A functions through induction of inflammatory mediators in both hematopoietic and nonhematopoietic compartments (19). Thus, we used the DR4tg mouse model, which provides a powerful in vivo system in which to delineate the role of early IL-17A responses in TSS. We treated DR4tg mice with an anti–IL-17A mAb 3 h prior to and 1 h after the administration of SEB and monitored disease progression. Neutralizing IL-17A at the TSS onset resulted in significantly attenuated weight loss as early as 20 h after SEB injection (Fig. 6A). Importantly, by 7 d after SEB injection, 67% of mice that were treated with anti–IL-17A, compared with only 27% of mice receiving the isotype control, had survived (Fig. 6B).

FIGURE 6.

IL-17A neutralization in a humanized mouse model of TSS reduces morbidity, mortality, and tissue damage. DR4tg mice were injected i.p. with 200 μg anti–IL-17A neutralizing Ab or isotype control 3 h before and 1 h after receiving 100 μg SEB. (A) Weight loss was monitored every 3 h for the first 12 h and every 8 h thereafter (n = 15 per group). Two-way ANOVA was used (p = 0.0012) with Holm–Sidak post hoc analysis. *p ≤ 0.05, **p ≤ 0.01. (B) Survival was monitored up to 7 d after SEB (n = 15 per group). Mice were sacrificed when moribund (≥20% weight loss). p = 0.0101, log-rank test. (C and D) At 48 h after SEB injection, mice were sacrificed for their organs and H&E staining was performed as per standard protocols. Sections were scored by a veterinary pathologist blinded to the experimental conditions. Histopathology scores represent sum of changes in the liver (periportal inflammation + microvesicular steatosis) and small intestine (inflammatory cell infiltration + mucosal score [epithelial cell damage + villar blunting]) observed in each section. Parameters received scores from 0 to 3, with 0 = normal, 1 = mild, 2 = moderate, and 3 = severe, and were analysed using two-tailed Student t test. Stained sections are representative of sham (n = 2), SEB + isotype control (n = 3) and SEB + anti–IL-17A (n = 4) groups. Mean ± SEM values are shown. (E) Two hours after SEB injection, anti–IL-17A– and isotype control–treated mice (n = 4 per group) were bled and the IL-6 content of serum samples was quantified by ELISA. Mean ± SEM values are shown. Two-way ANOVA was used (p = 0.05) with Holm–Sidak post hoc analysis. *p ≤ 0.05, **p ≤ 0.01.

FIGURE 6.

IL-17A neutralization in a humanized mouse model of TSS reduces morbidity, mortality, and tissue damage. DR4tg mice were injected i.p. with 200 μg anti–IL-17A neutralizing Ab or isotype control 3 h before and 1 h after receiving 100 μg SEB. (A) Weight loss was monitored every 3 h for the first 12 h and every 8 h thereafter (n = 15 per group). Two-way ANOVA was used (p = 0.0012) with Holm–Sidak post hoc analysis. *p ≤ 0.05, **p ≤ 0.01. (B) Survival was monitored up to 7 d after SEB (n = 15 per group). Mice were sacrificed when moribund (≥20% weight loss). p = 0.0101, log-rank test. (C and D) At 48 h after SEB injection, mice were sacrificed for their organs and H&E staining was performed as per standard protocols. Sections were scored by a veterinary pathologist blinded to the experimental conditions. Histopathology scores represent sum of changes in the liver (periportal inflammation + microvesicular steatosis) and small intestine (inflammatory cell infiltration + mucosal score [epithelial cell damage + villar blunting]) observed in each section. Parameters received scores from 0 to 3, with 0 = normal, 1 = mild, 2 = moderate, and 3 = severe, and were analysed using two-tailed Student t test. Stained sections are representative of sham (n = 2), SEB + isotype control (n = 3) and SEB + anti–IL-17A (n = 4) groups. Mean ± SEM values are shown. (E) Two hours after SEB injection, anti–IL-17A– and isotype control–treated mice (n = 4 per group) were bled and the IL-6 content of serum samples was quantified by ELISA. Mean ± SEM values are shown. Two-way ANOVA was used (p = 0.05) with Holm–Sidak post hoc analysis. *p ≤ 0.05, **p ≤ 0.01.

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Because TSS is characterized by multiorgan dysfunction, we investigated whether IL-17A neutralization affects SAg-induced liver and intestinal damage. DR4tg mice receiving SEB and either an anti–IL-17A mAb or an isotype control were sacrificed at 48 h post-SEB, and liver and the small intestine were collected for histopathological analyses. SEB induced moderate periportal inflammation in the livers of both anti–IL-17A– and isotype control–treated mice (Fig. 6C). However, hepatic steatosis, an early indication of hepatotoxicity (57), was greatly reduced in anti–IL-17A–treated mice compared with those receiving the isotype control (Fig. 6C). Furthermore, the small intestines of mice receiving anti–IL-17A showed less inflammation, epithelial cell damage, and villar blunting compared with the control cohort (Fig. 6C). As scored by a veterinary pathologist blinded to the cohorts, the overall TSS pathology for mice receiving anti–IL-17A was significantly reduced (Fig. 6D). Finally, we found a statistically significant decrease in the serum concentration of IL-6, a downstream target of IL-17A signaling, in anti–IL-17A–treated mice 2 h after SEB administration (Fig. 6E).

Collectively, to our knowledge, these results provide the first report that IL-17A is pathogenic in TSS and that neutralizing IL-17A ameliorates morbidity, mortality, and tissue damage in a humanized mouse model of TSS.

The inflammatory response mediated by IL-17A is the result of the induction and activation of a large network of immune effector molecules including cytokines, chemokines, acute phase reactants, tissue remodeling enzymes, antimicrobial peptides, and transcription factors (19). In this way, IL-17A plays a critical role in the immune response against many different pathogens including S. aureus (58) and S. pyogenes (59). However, unbalanced or dysregulated IL-17A responses may also lead to excessive inflammation and can therefore be detrimental in several diseased states (52). In this study, we report a pathogenic role for IL-17A during infection.

Much of the existing knowledge of TSS pathogenesis has relied on conventional mouse models (12). However, SAgs exhibit poor affinity for certain mouse MHC class II molecules (34), and as a result, many strains of laboratory mice are resistant to TSS. Sensitizing agents such as d-galactosamine (60) or LPS (61) have been employed to amplify the effects of SAgs and induce lethality, but the extent to which these models accurately reflect human TSS is questionable. For example, d-galactosamine pretreatment leads to extreme sensitivity to TNF-α–induced apoptosis in hepatocytes (62), which causes rapid mortality solely due to fulminant hepatitis, which does not represent human TSS (63). Here, we investigated SAg-induced cytokine responses utilizing DR4tg mice that transgenically express the human MHC class II molecule HLA-DR4. This allows for high-affinity SAg–MHC class II interactions resulting in robust activation of T cells and a cytokine storm that, if left untreated, simulates the potentially catastrophic human TSS (3537).

We found that exposure of DR4tg mice to SEB resulted in swift production of several cytokines in serum, including the inflammatory cytokine IL-17A. Cytokines secreted in the early phase of the cytokine storm are especially important as they trigger endothelial cell damage that compromises vascular homeostasis and precipitates multiorgan dysfunction (64). Furthermore, rapid secretion of IL-17A not only has inflammatory effects on a wide variety of cell types throughout the body, it can directly induce and also synergize with other cytokines that are produced in the same timeframe. Other studies have found mouse IL-17A secretion in response to SAgs (36, 44, 65). IL-17 is also reportedly detectable in SEB-stimulated human PBMC cultures, albeit at relatively late time points (66, 67). Here, we demonstrate that very early IL-17A is of CD4+ TEM cell origin in mice and in human PBMC cultures. Furthermore, we show that the rapid induction of IL-17A, in addition to other cytokines, is dependent on T cell stimulation because the injection of DR4tg mice with a TCR-binding mutant form of SEB (SEBN23A) resulted in minimal cytokine secretion. We believe the observed presence of IFN-γ, at moderate levels, after SEBN23A exposure is likely the consequence of SEB engaging T cells that bear Vβ domains other than Vβ8.2 (29). Lastly, we extended our investigation to other SAgs and found that SpeA and SEA could also induce early IL-17A secretion. Therefore, we propose that rapid IL-17A secretion plays a pathogenic role in both staphylococcal and streptococcal TSS.

To identify the location of IL-17A production in DR4tg mice, we generated IL-17A–GFP/DR4tg bone marrow chimeric mice as well as D17 mice in which IL-17A expression can be quantified by GFP fluorescence. Using these models, we found an increase in the frequency of IL-17A+ cells and/or the signal intensity of IL-17A in several tissues, but chiefly among IELs and LPLs from the small intestine, upon exposure to SEB. Interestingly, although IL-17A was consistently absent from the serum of PBS-injected mice, IL-17A–producing cells were readily detectable in these animals, suggesting that IL-17A is expressed at basal levels in multiple organs. This observation is in agreement with those of previous investigations utilizing alternatively generated IL-17A–GFP mice (68) or IL-17A fate-mapping reporter mice (69) that showed resting levels of IL-17A among cells of the small intestine. We expand on these findings to implicate CD4+ TEM cells as the main source of constitutively expressed IL-17A in naive mice. Nevertheless, it stands to reason that additional sources/locations of IL-17A may also exist.

Memory T cells are highly enriched in the intestinal mucosa and their propensity for immediate effector function upon reactivation (70) puts them in a unique position to be rapidly activated by SAgs. It has also been suggested that SAgs produced in the intestinal tract by food-borne pathogens may cross the gut mucosa directly (71), placing memory T cells among the first responders to SAgs. In support of this hypothesis, IL-17A was preferentially induced in the intestinal epithelial layer by CD4+ TEM cells when mice were exposed to SEB. Furthermore, a study by Tilahun et al. (36) demonstrated that most NF-κB expression in SEB-injected HLA-DR3 mice was induced in the intestines.

The IL-17A response to SEB in mice appears to be translatable to humans, as PBMCs from healthy human volunteers stimulated with SAgs in vitro also exhibited rapid IL-17A production. IL-17A was secreted within hours of SAg exposure along with other inflammatory cytokines such as IFN-γ, TNF-α, and IL-6. This inflammatory profile was most pronounced within the first few hours of SAg stimulation, with anti-inflammatory cytokines IL-10 and IL-13 only reaching appreciable levels toward later time points. This pattern of expression provides additional evidence suggesting that potential therapies aimed at curbing the overwhelming cytokine storm in TSS may be most effective early after exposure to SAgs. A study by Krakauer et al. (72) supports this line of reasoning, where mice surviving TSS showed reduced inflammatory cytokine and chemokine levels, though not including IL-17A, at 8 h post-SEB administration. Furthermore, in a TSS model utilizing HLA-DR1 mice, lethality was prevented by anti–TNF-α treatment, which dampened the early but not the later phase of cytokine release (73). Therefore, it is likely that the early phase of the cytokine storm is responsible for TSS lethality and that attenuating this response may be a fruitful strategy to prevent mortality.

Unexpectedly, we observed apparently paradoxical differences between the kinetics of mRNA upregulation and resulting protein expression between IL-17A and IFN-γ responses in SAg-stimulated PBMCs. We propose that the variation of kinetics between the two cytokines may be due, at least in part, to differences in mRNA stability. However, the mechanisms of posttranscriptional regulation of IL-17A remain ill-defined. In mice, the RNA-binding protein Hu Ag R positively regulates IL-17A expression and mRNA stability (74). Conversely, another RNA-binding protein, tristetraprolin, binds directly to the 3′ untranslated region of IL-17A mRNA to mediate its decay (75). Lastly, the positive correlation between microRNAs and IL-17A expression in many diseases other than TSS also suggests that microRNAs may play a critical role in regulating IL-17A responses (76). Whether RNA-binding proteins or microRNAs posttranscriptionally regulate human IL-17A in TSS remains to be elucidated.

Our findings indicate that CD4+ TEM cells predominate in the early production of both IL-17A and IFN-γ in SAg-stimulated human PBMCs. We also observed that IL-17A and IFN-γ responses by CD4+ TEM cells were mutually exclusive, with only a small number of cells capable of producing both cytokines simultaneously. Thus, we propose that SAgs stimulate pre-existing and independent lineage-committed CD4+ TEM cell subsets to rapidly produce IL-17A (TEM-17 cells) and IFN-γ (TEM-γ cells) in the initial phases of the cytokine storm. Our results may extend the traditional paradigm of Th1-, Th2-, and Th17-lineages of activated effector CD4+ T cells, which proceed to generate the body’s long-lived pool of TEM cells (77). This notion is supported by recent reports that human Th17 cells are a terminally differentiated TEM cell population with a high capacity of self-renewal (78, 79).

The timing and the intensity of SAg stimulation in human PBMCs also apparently affect the phenotype and cytokine profiles of activated cells. We found that at 4 h post-SEB stimulation, IL-17A–producing CD4+ TEM cells exclusively expressed the Th17-lineage transcription factor RORγT but did not coproduce IFN-γ. In contrast, a recent study by Björkander et al. (80) demonstrated that 24-h stimulation of human PBMCs with SAgs induces IL-17A in CD4+ T cells that express the regulatory T cell transcription factor FOXP3. As FOXP3 has been previously shown to be upregulated in SAg-stimulated CD4+ T cells over days of SAg exposure (81), it remains to be seen whether early IL-17A–producing CD4+ TEM cells may acquire FOXP3 expression after longer periods of SAg stimulation. In a separate study, overnight stimulation of human PBMCs with 10 μg/ml SEB (a 100-fold greater concentration of SAg than the dose used in this study) induced multifunctional CD4+ TEM cells that often coexpressed IL-17A and IFN-γ (51). Both at 4 and 24 h after SAg stimulation, we detected only a minute subpopulation of coproducing CD4+ TEM cells. It is possible that the high concentration of SAg used by McArthur et al. (51) may have been responsible for the coproducer phenotype. Circulating levels of SAg in patients’ serum samples range from ∼40 pg/ml in intensive care units (82) to 50–100 ng/ml in adult burn patients (83), and as high as 1000 ng/ml in streptococcal TSS patients (84). As such, our findings utilizing a more conservative concentration of SAg may represent a more physiologically relevant outcome for SAg stimulation of human PBMCs.

We found that the blockade of IL-17RA signaling in SEB-stimulated PBMCs attenuated the expression of multiple inflammatory genes including IL-6, TNF-α, and IFN-γ, which mediate many toxic effects of TSS (12). Concomitantly, the expression of anti-inflammatory cytokine genes (IL-10, TGF-β1, and IL-4) was upregulated by IL-17RA blockade. To our surprise, CCL20, a chemokine that binds CCR6 to mobilize lymphocytes and dendritic cells, was also upregulated. Nevertheless, the overall picture emerging from our IL-17RA blockade experiments strongly suggests a proinflammatory role for IL-17RA signaling in the context of human PBMC responses to SAgs. Equally important, IL-17RA blockade in dermal fibroblasts prevented their secondary response to SEB-exposed PBMCs. Therefore, this approach may work at multiple levels and may have therapeutic potentials for TSS. When patients with severe plaque psoriasis were treated with brodalumab, a mAb against IL-17RA, reduced expression of inflammatory cytokine transcripts was noted (85). Our results are also supported by the findings of Crowe et al. (86) that IL-17RA knockout mice show greatly attenuated cytokine storm when infected with influenza virus. In this model, the absence of IL-17RA signaling decreased lung inflammation, weight loss, and morbidity.

Remarkably, in vivo neutralization of IL-17A in DR4tg mice at the onset of TSS led to a significant reduction in weight loss and a striking increase in survival. We also observed drastic pathological changes in both the liver and the epithelium of the small intestine in DR4tg mice exposed to SEB. These mice developed hepatic microvesicular steatosis, an early toxic change in hepatocytes that signals serious disease. Characterized by the accumulation of small lipid vesicles in hepatocytes, microvesicular steatosis is indicative of impaired mitochondrial β-oxidation of fatty acids and can lead to multiorgan dysfunction (57). Importantly, microvesicular steatosis occurs in both human TSS (87, 88) and sepsis (89). To our knowledge, our work is the first description of its reversal in an animal model of TSS. We also observed considerable epithelial damage and villar blunting in the small intestine of mice injected with SEB. These pathological changes likely impair intestinal function and contribute to the weight loss observed in SAg-exposed mice. Importantly, in vivo neutralization of IL-17A in this model markedly attenuated both hepatic steatosis and damage to the intestinal epithelium. As almost all human TSS patients show multiorgan dysfunction (90), our results suggest that IL-17A may be an attractive clinical target to reduce TSS severity.

The key to successful treatment of TSS remains the rapid identification of the source of infection, appropriate and effective use of antibiotics, drainage of wounds (or removal of tampon in menstrual TSS), and supportive care (91). IVIg has also been shown to improve clinical outcomes in TSS caused by group A Streptococcus (92). However, there is currently no cure for the cytokine storm induced by SAgs. Here, we define the rapid production of IL-17A by CD4+ TEM cells as a novel mechanism underlying TSS immunopathology. Our findings suggest that neutralizing IL-17A function may serve to lessen systemic inflammatory cytokine levels and decrease tissue damage in TSS. Thus, a therapeutic approach targeting the early induction of multiple inflammatory mediators that are involved in the cytokine storm, including IL-17A, may be effective in averting TSS mortality.

We thank Dr. John Koval (Department of Epidemiology and Biostatistics, Western University) for expert statistical advice. We thank members of the Haeryfar laboratory for helpful discussions and Madeline Harvey for assistance with mouse irradiation.

This work was supported by a Canadian Institutes of Health Research operating grant (Grant MOP-130465) to S.M.M.H.

The online version of this article contains supplemental material.

Abbreviations used in this article:

act-D

actinomycin D

CEBPD

C/EBP δ

CT

cycle threshold

D17

HLA-DR4 transgenic IL-17A reporter

DR4tg

HLA-DR4 transgenic

IEL

intraepithelial lymphocyte

IL-17RA

IL-17 receptor A

iNKT

invariant NKT

LPL

lamina propria lymphocyte

MAIT

mucosa-associated invariant T

MFI

mean fluorescence intensity

qPCR

quantitative real-time PCR

RPL13A

ribosomal protein L13a

SAg

superantigen

SEA

staphylococcal enterotoxin A

SEB

staphylococcal enterotoxin B

SmeZ

streptococcal mitogenic exotoxin Z

SpeA

streptococcal pyrogenic exotoxin A

SpeI

streptococcal pyrogenic exotoxin I

TEM

effector memory T

TSS

toxic shock syndrome

TSST-1

TSS toxin-1.

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The authors have no financial conflicts of interest.

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