Response Enterotoxin a Lung Staphylococcus Aureus Induced By

CD169+ Macrophages Restrain Systemic Inflammation Induced by Staphylococcus aureus Enterotoxin A Lung Response Downloaded from Julia Svedova, Antoine Ménoret, Stephen T. Yeung, Masato Tanaka, Kamal M. Khanna and Anthony T. Vella

ImmunoHorizons 2017, 1 (9) 213-222

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Adaptive Immunity

CD169+ Macrophages Restrain Systemic Inflammation Induced by Staphylococcus aureus Enterotoxin A Lung Response

Julia Svedova,* Antoine Menoret,*´ ,† Stephen T. Yeung,* Masato Tanaka,‡ Kamal M. Khanna,* and Anthony T. Vella* *Department of Immunology, School of Medicine, University of Connecticut Health Center, Farmington, CT 06030; †Institute for Systems Genomics, University of Connecticut Health Center, Farmington, CT 06030; and ‡School of Life Science, Tokyo University of Pharmacy and Life Downloaded from Sciences, Tokyo 192-0392, Japan

ABSTRACT http://www.immunohorizons.org/ Alveolar macrophages (AMs) are considered the first line of defense in the airways. Exposure to harmful substances and certain infections can lead to dysfunction or depletion of AMs. Importantly, these conditions have been associated with increased risk of sepsis and acute lung injury. Staphylococcus aureus enterotoxins are superantigens that induce oligoclonal activation of T cells and a robust cytokine release, leading to systemic inflammatory response and tissue injury. In this study we investigated the relationship between S. aureus enterotoxins and AMs. Following inhalation, S. aureus enterotoxin was preferentially bound to AMs and MHC class II was not required. Furthermore, the enterotoxin was internalized and its presence in the cells decreased by 24 h after exposure. Ablation of AMs in CD169–diphtheria toxin receptor mice was associated with increased activation of enterotoxin-specific T cells and enhanced cytokine release into circulation. Thus, conditions causing depletion of AMs may increase the risk of S. aureus enterotoxin–induced diseases. ImmunoHorizons, 2017, 1: 213–222. by guest on September 26, 2021 INTRODUCTION models, including influenza, asthma, and acute lung injury (2–4), but they could be also proinflammatory (5). Alveolar macrophages (AMs) are specialized tissue-resident AMs are a relatively stable population of cells with very slow cells that play a crucial role in lung development, homeostasis, turnover rate; however, when the injury is severe enough, AMs and immune surveillance. Their unique position in the lung may be depleted and later replenished from circulating monocytes alveoli enables them to sample pathogens, inhaled particulates, or from in situ proliferation (1). Exposure to various substances, and other environmental cues. Their reciprocal interactions pathogens, and their products may lead to AM depletion or im- with neighboring cells through various receptors and the paired function; these include influenza (6), bacterial pneumo- cytokine/chemokine axis can then alarm the immune system nia (7), cigarette smoke (8), anesthesia (9), diabetes (10), or alcohol and orchestrate responses to remove potential threats and consumption (11). Importantly, many of these conditions or respond to tissue injury, making them the first line of pul- exposures have been associated with increased risk of serious monary immune defense (1). AMs have been shown to play a infections and development of systemic inflammatory response critical anti-inflammatory role in a number of disease animal syndrome/sepsis and acute respiratory distress syndrome (ARDS).

Received for publication July 21, 2017. Accepted for publication October 27, 2017. Address correspondence and reprint requests to: Dr. Anthony T. Vella, Department of Immunology, School of Medicine, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030. E-mail address: [email protected] ORCIDs: 0000-0003-0836-4725 (J.S.); 0000-0002-9710-5567 (S.T.Y.); 0000-0002-9328-3817 (K.M.K.). A.T.V. and J.S. conceived and designed the study; A.T.V. and J.S. analyzed the data; J.S., A.T.V., and A.M. drafted the manuscript; J.S., A.M., and S.T.Y. performed experiments; and M.T. and K.M.K. provided reagents and expertise and reviewed the manuscript. This work was supported by National Institute of Allergy and Infectious Diseases Grant P01AI056172. Abbreviations used in this article: AM, alveolar macrophage; ARDS, acute respiratory distress syndrome; BAL, bronchoalveolar lavage; DT, diphtheria toxin; DTR, diphtheria toxin receptor; MHC II, MHC class II; WT, wild-type. The online version of this article contains supplemental material. This article is distributed under the terms of the CC BY-NC-ND 4.0 Unported license. Copyright © 2017 The Authors https://doi.org/10.4049/immunohorizons.1700033 213

ImmunoHorizons is published by The American Association of Immunologists, Inc. 214 CD169+ CELLS ATTENUATE S. AUREUS ENTEROTOXIN RESPONSE ImmunoHorizons

In particular, influenza can be complicated by a secondary bacte- B alone (31). Based on these findings, we hypothesized that AMs rial pneumonia, which leads to higher rates of hospitalization and play a critical role in the inflammatory responses triggered by death (12). Increased alcohol consumption and cigarette smoke S. aureus enterotoxin inhalation. exposure have been associated with increased risk of systemic In this study we show that enterotoxin A was taken up by AMs inflammatory response syndrome/septic shock and ARDS, re- upon inhalation and the enterotoxin was found intracellularly and spectively (13, 14). Finally, a number of chronic conditions, partic- not on the cell surface. Perhaps surprisingly, MHC II expression ularly diabetes mellitus, and immunosuppressive state are well- was not required for binding, as enterotoxin A was similarly taken described comorbidities in sepsis patients (15, 16). Collectively, up by AMs of wild-type (WT) and MHC II2/2 mice. AMs in the these studies suggest that depletion or impaired function of AMs murine lung express sialoadhesin, also known as CD169, on their may be associated with increased risk of serious infection, sepsis, surface (32). Thus, to study the role of AMs, CD169–diphtheria and ARDS. toxin receptor (DTR) mice were treated with DT and then exposed Staphylococcus aureus enterotoxins are a group of potent to enterotoxin A. The absence of AMs was associated with bacterial toxins that bypass the classical Ag processing and increased CD25 expression on enterotoxin A–specificTcells,but presentation, directly bind to MHC class II (MHC II) on APCs, this effect was only observed when lower concentrations of and crosslink it with specificVb-chains of TCRs. This unique enterotoxin A were administered. Finally, enterotoxin A–exposed Downloaded from feature of the so-called superantigens induces a massive in- CD169-DTR mice also showed a significantly greater cytokine flammatory response marked by oligoclonal T cell activation and secretion to blood compared with WT mice. Thus, AMs may cytokine storm (17). Superantigens have been established as the represent a defense mechanism against S. aureus enterotoxins, as mediators of toxic shock syndrome, a type of systemic in- their depletion may increase the severity of toxic shock or other flammatory response, which can lead to tissue injury, shock, and S. aureus enterotoxin–induced diseases. http://www.immunohorizons.org/ even death (18). However, recent evidence suggests that they may be involved in a number of diseases, including pneumonia, endo- carditis, and sepsis (19–21). In fact, superantigens and superantigen- MATERIALS AND METHODS expressing S. aureus strains have been recovered from septic patients (22). S. aureus enterotoxins likely spread systemically Mice from focal sites of infection rather than being directly produced in C57BL/6J (WT) and MHC II2/2 mice were purchased from The the bloodstream, as hemoglobin peptides were shown to inhibit Jackson Laboratory (Bar Harbor, ME) and CD169-DTR mice superantigen production (19). The most common colonization site (CD169-DTR+/2) were provided by Dr. K.M. Khanna (University of S. aureus is the anterior nares (23); therefore, exposure to of Connecticut Health Center via Dr. M. Tanaka). Mice were used aerosolized or inhaled S. aureus enterotoxin may well mimic the between1.5and4moofageandtheywerehousedintheCentral by guest on September 26, 2021 route of dissemination. Accidental exposure to aerosolized Animal Facility at the University of Connecticut Health Center S. aureus enterotoxin in humans was reported to cause systemic according to federal guidelines. All experimental procedures were symptoms of fever, chills, headache, myalgia, cough, dyspnea, approved by the Institutional Animal Care and Use Committee at vomiting, and diarrhea in humans (24). In nonhuman primates, the University of Connecticut Health Center. aerosolized lethal doses of S. aureus enterotoxin caused severe pulmonary lesions and death in some subjects (25). Finally, Toxin administration S. aureus enterotoxin inhalation in mice was shown to cause In most studies, 1 mg of enterotoxin A (Toxin Technology, Sarasota, systemic inflammatory response, involving rapid activation of FL) diluted in 50 ml of balanced salt solution or balanced salt T cells, cytokine release, recruitment of innate cells, and subse- solution alone (vehicle) was pipetted on the nostrils (intranasal quent lung injury (26). route). In enterotoxin A titration experiments, 0.033, 0.1, 0.33, or The relationship between AMs and S. aureus enterotoxins has 1 mg of enterotoxin A was administered intranasally. To ablate not been fully explored. In vitro stimulation of human AMs with S. CD169+ cells, both WT and CD169-DTR mice received 40 ng/g aureus enterotoxin A (enterotoxin A) induced IL-8 production in a body weight of DT (Sigma-Aldrich, St. Louis, MO) i.p. 2 d before dose-dependent manner (27). In another in vitro study, addition enterotoxin A or vehicle inhalation. of macrophages (AMs or peritoneal macrophages) reduced proliferation of enterotoxin A–activated T cells via NO production Flow cytometry (28). Although there is only a limited direct link between AMs and Harvest and processing of bronchoalveolar lavage (BAL) fluid, S. aureus enterotoxins, indirect evidence exists that shows a lung, and spleen, and staining for flow cytometry were performed relationship between superantigens and conditions that deplete as described previously (26, 33). The single-cell suspensions were AMs. In particular, several reports from the 1980s described labeled with Live/Dead fixable blue dead cell stain (Thermo Fisher development of toxic shock syndrome in patients with influenza Scientific, Waltham, MA) and stained with the following Abs: and influenza-like illness (29, 30). Furthermore, a recent study CD45.2 clone 104 (shown as CD45), anti–Siglec F clone E50-2440, showed that the combination of cigarette smoke exposure and anti-CD11c clone N418, anti-CD169 clone SER-4, anti-CD3 S. aureus enterotoxin B (enterotoxin B) induced a greater extent of clone 17A2, anti-Vb3 clone KJ25, anti-Vb14 clone 14-2, anti-CD25 pulmonary inflammation than did cigarette smoke or enterotoxin clone PC61, and anti-CD69 clone H1.2F3 (purchased from BD

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Biosciences, San Jose, CA or eBioscience, San Diego, CA). Staining lymphoid tissues within minutes of inhalation (26, 34). Further- for enterotoxin A was performed (after blocking with undiluted more, using a titration curve, the concentration of enterotoxin A in goat serum) with polyclonal anti–enterotoxin A Ab produced in serum after inhalation was estimated to be only a small fraction of rabbit (whole antiserum; Sigma-Aldrich) followed by secondary the total amount inhaled by a mouse (;,1/35th; J. Svedova, goat anti-rabbit IgG–Alexa Fluor 488 (Thermo Fisher Scientific). unpublished observations). Intriguingly, in another report using For intracellular staining, cells were first fixed in 2% formaldehyde mass spectrometry, we showed that enterotoxin A could be and then permeabilized with 0.25% saponin (Sigma-Aldrich). Flow recovered from BAL fluid at 16 h after inhalation and the toxin cytometry was performed using a FACS LSR II (BD Biosciences) preserved its biological activity (35). These findings suggested that and the data were analyzed with FlowJo software (Tree Star, when enterotoxin A is inhaled, only a small portion enters the Ashland, OR). circulation, whereas the remainder may be retained in the lung mucosa or perhaps neutralized by various defense mechanisms, Confocal microscopy such as defensins or phagocytosis. Confocal microscopy on frozen tissue sections was performed as Therefore, we sought to determine whether enterotoxin A described before (26). The sections were stained with polyclonal binds to a certain cell population within the pulmonary tissue. anti–enterotoxin A Ab (Sigma-Aldrich) for 1 h at room tempera- Lung was obtained from mice 8 h after enterotoxin A or vehicle Downloaded from ture, which was followed by Alexa Fluor 488–conjugated anti- inhalation and examined by confocal microscopy for enterotoxin A rabbit IgG (Thermo Fisher Scientific) and fluorophore-conjugated using polyclonal anti–enterotoxin A Ab. Only mice exposed to Abs for 1 h at room temperature. The following Abs were used: enterotoxin A displayed cells that were enterotoxin A+ (Fig. 1A, anti-CD169 clone SER-4, MHC II clone M5/114.15.2, and anti- 1B, Supplemental Fig. 1A), confirming the specificity of the Ab. CD11c clone N418 or HL3 (eBioscience and BD Biosciences). The Surprisingly, we found that enterotoxin A was preferentially http://www.immunohorizons.org/ images were acquired with a Zeiss LSM780 confocal microscope bound to CD11c+CD169+ AMs (Fig. 1A). In fact, a total of 87% of (Zeiss, Oberkochen, Germany) as confocal z-stack projections, enterotoxin A+ cells were also positive for CD11c and 64% were using 320/0.8 numerical aperture objective. Image analysis, both CD11c+ and CD169+ (Fig.1C).Tofurtherdefine enterotoxin A including adjustment for brightness and contrast, quantification of binding to AMs, BAL fluid was obtained from mice 1, 4, or 24 h cells, and colocalization studies (threshold value set to 7), was after enterotoxin A inhalation and BAL AMs defined as CD45+ performed with Imaris software (Bitplane, Zurich, Switzerland). CD11c+Siglec F+CD169+ (Fig. 1D) were stained for enterotoxin A. Importantly, no enterotoxin A was detected on the surface of Multiplex assay AMs; however, there was a significant increase in enterotoxin A Serum was collected using serum separator tubes (BD Biosci- presence intracellularly, peaking at 4 h (Fig. 1E, 1F, Supplemental ences). The concentrations of cytokines in the serum were mea- Fig. 1B). Interestingly, there was no significant difference detected by guest on September 26, 2021 sured with customized Luminex-based multiplex assays (EMD at 24 h after enterotoxin A (Fig. 1F). Similar results were obtained Millipore, Billerica, MA). when AMs from lung tissue were analyzed (Fig. 2A). In contrast, lung dendritic cells showed increased enterotoxin A expression Statistical analysis only at 24 h both on the surface and intracellularly (Fig. 2). Statistical analyses in all experiments were performed using Prism S. aureus enterotoxins are known for their ability to directly (GraphPad Software, La Jolla, CA) and Microsoft Excel. A bind to MHC II molecules and crosslink them with specificVb- p value , 0.05 was considered significant (*p , 0.05, **p , 0.01, chains of TCRs, leading to a systemic inflammatory response (17). ***p , 0.001). However, AMs were reported to only express low levels of MHC II under homeostatic conditions (1) with MHC II expression increasing during an inflammatory response, particularly in the RESULTS presence of IFN-g (36). Furthermore, AMs tend to be poor Ag presenters and may, in fact, inactivate T cells (37). These findings MHC II in lung is not required for binding of enterotoxin A suggested that enterotoxin A binding to AMs can occur inde- to AMs pendently of MHC II expression. To assess whether enterotoxin One of the crucial properties of S. aureus enterotoxins is their A requires MHC II for binding to AMs, lung tissue was harvested ability to rapidly induce oligoclonal activation of T cells, which is from WT and MHC II2/2 mice 8 h after enterotoxin A inhalation accompanied by a massive inflammatory response and cytokine and analyzed by confocal microscopy. There was no difference in storm (17). However, how enterotoxin A and other enterotoxins the number of enterotoxin A+ cells between WT and MHC II2/2 spread systemically, especially from the airways, as anterior nares mice (Fig. 3A, 3B). Additionally, enterotoxin A+ cells colocalized are a common site of S. aureus colonies(23), isnotfully understood. with CD11c marker with a similar frequency (Fig. 3C). Finally, only In our previous studies, we showed that enterotoxin A permeates a small fraction of enterotoxin A+ cells colocalized with MHC II into blood immediately after inhalation and is found in the serum (Fig. 3D) and, in fact, we observed CD11c+MHC II+ dendritic cells rather than bound to cells (26). The ability to access the circulatory with no significant enterotoxin A binding (Fig. 3A, white arrows). system from airway mucosa is likely an important feature that Collectively, these results show that enterotoxin A binding to AMs enables the toxin to spread systemically and to activate T cells in does not require MHC II expression.

https://doi.org/10.4049/immunohorizons.1700033 216 CD169+ CELLS ATTENUATE S. AUREUS ENTEROTOXIN RESPONSE ImmunoHorizons Downloaded from http://www.immunohorizons.org/ by guest on September 26, 2021 FIGURE 1. Enterotoxin A preferentially binds to AMs upon inhalation. (A) Representative confocal microscopy images of lung tissue 8 h after enterotoxin A or vehicle inhalation. Lung sections were stained with anti–enterotoxin A (green), anti-CD11c (red), and anti-CD169 (blue). Magnified images of the areas marked by white squares in the overlay panels are shown on the left. Scale bars, 80 mm. Four independent experiments with a total of n = 5 per group were used. (B) The number of enterotoxin A+ cells per field. Three images from each mouse were quantified (four independent experiments with n = 5 per group) by Imaris (Bitplane). (C) Percentage of CD11c+CD169+ and CD11c+CD1692 cells in all enterotoxin A+ cells detected by confocal microscopy 8 h after enterotoxin A inhalation. Enterotoxin A+ cells were colocalized with CD11c+ cells and enterotoxin A+ CD11c+ cells were further colocalized with CD169 by Imaris. The average percentage shown in the pie chart was calculated from three images per mouse (three independent experiments with total n = 4). (D) Gating strategy to identify AMs in the BAL fluid by flow cytometry. BAL fluid was obtained 1, 4, or 24 h after enterotoxin A inhalation or from vehicle control (1 or 4 h) and AMs were identified as live CD45+CD11c+Siglec F+CD169+ cells. (E) Representative histograms showing enterotoxin A expression on surface and intracellularly plus surface in BAL fluid AMs. (F) Relative median fluorescence intensity (MFI) of enterotoxin A on surface and intracellularly plus surface in BAL fluid AMs 1, 4, or 24 h after enterotoxin A inhalation. MFI values are relative to the vehicle control (set as 1). Three independent experiments with a total of n = 7 per group were performed. Data are represented as mean 6 SEM. An unpaired t test (B) or one- way ANOVA with a Dunnett test (F) were used: *p , 0.05, ***p , 0.001.

Depletion of AMs is associated with increased T cell DT administration. Because AMs express CD169 (Fig. 1), this activation and cytokine secretion technique can be used to study the role of AMs in pulmonary Next, we wanted to determine whether enterotoxin A binding to responses (32). Thus, CD169-DTR mice and WT control were AMs impacts T cell activation and cytokine production. We treated with DT 2 d prior to enterotoxin A or vehicle inhalation. To hypothesized that AMs sequester a large portion of the inhaled avoid oversaturation of enterotoxin A dosage given to mice (which toxin and neutralize it. Thus, AMs could play a protective role in couldleadtomaskingofdifferences between WT and CD69-DTR enterotoxin A–induced inflammation. To investigate the role of mice), the enterotoxin A dose was titrated (0.033, 0.1, 0.33, and AMs in enterotoxin A–induced inflammatory response, we used 1 mg). Blood, lung, and spleen were harvested 4 h after enterotoxin CD169-DTR mice to selectively deplete CD169+ macrophages after A or vehicle (0 mg) inhalation. Depletion of CD169+ AMs was

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in CD25+Vb14+ T cells in CD169-DTR mice after a dose of 0.033 mg of enterotoxin A (Fig. 4D, 4F, right panels). A hallmark of toxic shock syndrome as well as sepsis is a robust release of cytokines into the bloodstream (18). Therefore, we next investigated whether the depletion of CD169+ macrophages affected cytokine secretion following enterotoxin A inhalation. Serum cytokine concentrations were measured by a multiplex assay in DT-pretreated WT and CD169-DTR mice 4 h after vehicle or enterotoxin A (0.033 or 0.1 mg) inhalation. There was no significant difference in the levels of serum cytokine when vehicle control was administered. However, following enterotoxin A inhalation, CD169-DTR mice had greater concentrations of serum G-CSF, IFN-g, TNF, IL-2, IL-6, and also IL-10 compared with WT mice (Fig. 5). These findings show that ablation of AMs is associated with increased expression of CD25 on enterotoxin Downloaded from A–specificVb3+ T cells and also increased secretion of cytokines into the circulation.

DISCUSSION http://www.immunohorizons.org/

Superantigens such as S. aureus enterotoxins are potent bacterial FIGURE 2. Enterotoxin A presence in lung AMs and dendritic cells. toxins that can induce serious inflammatory responses and lethal Lung tissue was harvested 1, 4, or 24 h after enterotoxin A inhalation or shock (17). It has been estimated that most strains of S. aureus are from vehicle control (1 or 4 h). AMs were identified as live CD45+CD11c+ capable of producing superantigens (19). However, S. aureus is also Siglec F+CD169+ cells and dendritic cells as live CD45+CD11c+Siglec an extremely common commensal that permanently colonizes F2MHC II+.(A) Relative median fluorescence intensity (MFI) of entero- ;20% of all individuals (23). Thus, there is a disparity in under- toxin A on surface and intracellularly plus surface in lung AMs 1, 4, or standing why S. aureus is so common in asymptomatic individuals 24 h after enterotoxin A inhalation. (B) Relative MFI of enterotoxin A on and, simultaneously, it can trigger a life-threatening inflammatory surface and intracellularly plus surface in lung dendritic cells 1, 4, or response. Although the mechanisms of how a commensal coloni- by guest on September 26, 2021 24 h after enterotoxin A inhalation. MFI values are relative to the ve- zation can become pathogenic are not clear (38), it has been well hicle control (set as 1). Two independent experiments with a total of established that patients who have colonization of anterior nares n = 4 per group were performed. Data are represented as mean 6 SEM. with S. aureus and particularly methicillin-resistant S. aureus are One-way ANOVA with a Dunnett test was used: *p , 0.05, **p , 0.01, more susceptible to hospital-acquired S. aureus infections (39). ***p , 0.001. This suggests that various defense mechanisms must be in place to prevent the spread of the bacteria and limit the effects of the confirmedinthelungbygatingonCD45+CD11c+Siglec F+CD169+ virulence factors; however, these mechanisms may be impaired in cells (Fig. 3A). The overall depletion of AMs was ;87% (Fig. 3B). immunocompromised individuals in hospital settings. We first investigated the effect of CD169+ macrophage ablation Indeed, the immune system possesses several strategies to on CD69 expression on enterotoxin A–specificVb3+ Tcellsin counteract the harmful effects of superantigens. First, it has been the spleen. Interestingly, even with a low dose of enterotoxin A hypothesized that the production of Abs against superantigens is (0.033 mg), the percentage of CD69+Vb3+ Tcellswas88.5%inWT protective and the lack of Abs may a predisposing factor for the mice and 89.8% in CD169-DTR mice, suggesting that a very small development of S. aureus infections and toxic shock syndrome amount of enterotoxin A is sufficient to induce CD69 expression (40). In particular, women with toxic shock syndrome had lower on T cells (Fig. 4C, 4D). Thus, we concluded that CD69 expres- Ab titers to TSST-1 compared with healthy women with no prior sion might not be a sensitive marker to detect differences in T cell history of toxic shock syndrome (41). Similarly, immunotherapy activation. In contrast, CD25 expression on Vb3+ T cells increased using Abs specific to superantigens was found to be protective in in a dose-dependent manner (Fig. 4F). Importantly, CD169-DTR animal models (42). In addition to Abs, it has been shown that mice showed significantly greater percentage of CD25+Vb3+ lactoferrin, an iron-binding glycoprotein located predominantly in T cells when administered 0.033 and 0.1 mg of enterotoxin A mucosal secretions (43), can attenuate the inflammatory responses (Fig. 4E, 4F). There was no significant effect on CD25 expression triggered by S. aureus enterotoxins (35). In this study we show that with the higher doses of enterotoxin A (0.33 or 1 mg; Fig. 4F). We AMs may represent a key cellular defense mechanism that can also investigated the effect of AM ablation on the bystander Vb14+ reduce the effects of S. aureus enterotoxin–induced inflammation. T cells. There was no difference in the percentage of CD69+ and When staining for enterotoxin A in the lung tissue, we found CD25+ of Vb14+ T cells with the exception of a significant increase that AMs preferentially bound enterotoxin A after inhalation and

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FIGURE 3. MHC II is not required for binding of enterotoxin A to AMs. (A) Representative confocal microscopy images comparing enterotoxin A binding in WT and MHC II2/2 mice 8 h after enterotoxin A inhalation. Lung sections were stained with anti– enterotoxin A (green), anti-CD11c (red), and anti-MHC II (blue). Magnified images of the areas marked by white squares in the overlay panels are shown on the left. White arrows in the magnified panel (WT) point to dendritic cells (CD11c+MHC II+). Scale bars, 80 mm. Three independent experiments with a total of n = 5 per group were performed. (B) Number of enterotoxin A+ cells per field. Three images by guest on September 26, 2021 from each mouse were quantified (three independent experiments with n = 5 per group) by Imaris (Bitplane). (C) Percentage of CD11c+ cells in all enterotoxin A+ cells detected by confocal microscopy 8 h after enterotoxin A inhalation. Enterotoxin A+ cells in WT and MHC II2/2 mice were colocalized with CD11c+ cells by Imaris (three images per mouse; three independent experiments with n = 5 per group). (D) Percentage of MHC II+ cells in all enterotoxin A+ cells detected by confocal microscopy 8 h after enterotoxin A inhalation. Enterotoxin A+ cells in WT mice were colocalized with MHC II+ cells by Imaris (three images per mouse; three independent experiments with n = 5 per group). Data are represented as mean 6 SEM. Unpaired t test. internalized it (Fig. 1). Interestingly, the intracellular presence of which results in rapid oligoclonal activation of T cells and a enterotoxin A in AMs increased in the first several hours but was cytokine storm (17). Additionally, compared with enterotoxin B, not significantly different at 24 h (Figs. 1F, 2A). Additionally, enterotoxin A was found to have a greater affinity for MHC II due dendritic cells, which were hypothesized to bind a significant to its ability to not only bind the a-chain but also the b-chain of amount of enterotoxin A due to their high MHC II expression, MHC II with a zinc-dependent binding site, which enabled showed upregulation of enterotoxin A expression on surface and enterotoxin A to stay on the cell surface for at least 40 h (46). intracellularly only at 24 h after inhalation (Supplemental Fig. 1B). Interestingly, we found that the binding of enterotoxin A to AMs Based on these findings, we postulate that the numerous and did not require MHC II expression (Fig. 3). Binding of S. aureus highly phagocytic AMs ingest enterotoxin A and thus reduce the enterotoxins to molecules other than MHC II have been previ- ability of dendritic cells either in the lung mucosa or in other ously reported. In particular, the costimulatory molecule CD28 tissues to take up the Ag and present it. In fact, AMs have been and its ligand CD86 are crucial binding sites that enhance the shown to maintain lung homeostasis by suppressing dendritic cell severity of the inflammatory response triggered by S. aureus en- function (44). The later presence of enterotoxin A on pulmonary terotoxin (47). Other binding sites for S. aureus enterotoxins have dendritic cells could perhaps be due to Ag transfer from AMs to been reported, including MHC class I (48), digalactosylceramide dendritic cells (45). on kidney proximal tubular cells (49), and Gp130 receptor on Superantigens, such as S. aureus enterotoxins, are known for adipocytes (50). These studies show that S. aureus enterotoxins their ability to bind to MHC II directly without Ag processing, may bind to several different molecules. Thus, it will be important

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FIGURE 4. Depletion of CD169+ cells leads to increased expression of CD25 on enterotoxin A–specific Vb3+ T cells. WT and CD169-DTR mice received DT 2 d prior to enterotoxin A or vehicle inhalation. Lung and spleen were removed 4 h after vehicle (0 mg) or enterotoxin A (0.033, 0.1, 0.33 or 1 mg) inhalation. (A and B) Representative flow cytometry plots (A) and scatter dot plot (B) show depletion of AMs in lung (gated as live CD45+CD11c+Siglec F+CD169+ cells) in CD169-DTR mice compared with WT mice. The percentage of AMs is relative to all live cells. (C) Representative histogram of CD69 expression in live CD45+CD3+Vb3+ cells in WT (black) and CD169-DTR (red) mice 4 h after 0.033 mgof enterotoxin A inhalation. Gray, isotype control. (D) Percentage of CD69+ cells in Vb3+ (left) and Vb14+ (right) T cells 4 h after 0, 0.033, 0.1, 0.33, or 1 mg of enterotoxin A inhalation. (E) Representative histogram of CD25 expression in live CD45+CD3+Vb3+ cells in WT (black) and CD169-DTR (red) mice 4 h after 0.033 mg of enterotoxin A inhalation. Gray, isotype control. (F) Percentage of CD25+ cells in Vb3+ (left) and Vb14+ (right) T cells 4 h after 0, 0.033, 0.1, 0.33, or 1 mg of enterotoxin A inhalation. The data were compiled from three independent experiments with a total of n =5–6 per group. A two-way ANOVA with a Sidak test was used: *p , 0.05, **p , 0.01, ***p , 0.001. to determine the identity of the binding site of enterotoxin A on AMs CD25 or CD69 on enterotoxin A–specificVb3+ T cells as well as no as well as the processing of the enterotoxin by these phagocytes. significant change in serum cytokines when mice were adminis- Previous studies showed that following intranasal S. aureus tered vehicle control (Figs. 4, 5). However, CD169-DTR exposed to enterotoxin exposure, mice experience a systemic oligoclonal enterotoxin A showed a significantly greater CD25 expression T cell activation and cytokine release to blood (26, 33, 51). To compared with WT mice (Fig. 4F). This was only observed when examine the role of AMs in the enterotoxin A–induced in- lower amounts of enterotoxin A were given, suggesting that the flammatory response, WT and CD169-DTR mice were treated higher doses of enterotoxin A saturate the level of T cell activation. with DT and 2 d later, they were exposed to vehicle or enterotoxin Indeed, even 0.033 mg of enterotoxin A activated almost 90% of A. There was no difference in the expression of activation markers enterotoxin A–specificVb3+ T cells in the spleen (Fig. 4F). These

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FIGURE 5. Depletion of CD169+ cells increases the concentration of serum cytokines after enterotoxin A inhalation. Serum was obtained from blood 4 h after vehicle (0 mg) or enterotoxin A (0.033 or 0.1 mg) inhalation. The concentrations of different cytokines were measured by a bead-based multiplex assay (EMD Millipore). The data were compiled from three independent experiments with a total of n = 6 per http://www.immunohorizons.org/ group. A two-way ANOVA with a Sidak test was used: *p , 0.05, **p , 0.01, ***p , 0.001.

findings emphasize the potency of enterotoxin A, which even in the subsequent cell apoptosis could play a role in the increased a small amount is sufficient to trigger a significant response. inflammatory response in CD169-DTR mice. Interestingly, the Furthermore, we examined the concentrations of serum cytokines absence of CD169+ macrophages during viral infection in mice commonly associated with toxic shock syndrome and sepsis, in reduces antiviral type 1 IFN activity and also limits PD-L1 expression, particular, TNF, IFN-g,IL-2,andIL-6(18,52).IL-10andG-CSF leading to overwhelming viral replication in the absence of CD8+ were also analyzed. G-CSF, a growth factor that mobilizes T cell exhaustion (57), a mechanism that could be considered in our neutrophils into circulation during acute inflammation (53), was model. Because of the broad expression of CD169 in secondary by guest on September 26, 2021 previously found elevated in patients with bacterial infection (54) lymphoid tissue and the emerging role of CD169+ macrophages in whereas the anti-inflammatory cytokine IL-10 can be released in inflammation and immunoregulation (58), further studies will be septic patients in an attempt to counteract the hyperinflammatory necessary to confirm the causal relationship between AMs and response (52). Importantly, enterotoxin A–exposed CD169-DTR S. aureus enterotoxin–induced systemic inflammatory response. mice had increased secretion of proinflammatory cytokines TNF, In conclusion, in this study we show that AMs bind S. aureus IFN-g, IL-2, and IL-6, as well as G-CSF, demonstrating that AM enterotoxin irrespective of MHC II expression and internalize it. ablation is associated with enhanced inflammatory response Furthermore, AM ablation in CD169-DTR mice was associated following enterotoxin A inhalation (Fig. 5). Interestingly, the with increased activation of enterotoxin A–specificVb3+ Tcells concentration of anti-inflammatory IL-10 was also elevated in and enhanced secretion of cytokines into circulation. These CD169-DTR mice (Fig. 5). This is consistent with our previous findings could explainthe development of toxic shock syndrome in study showing that enterotoxin A simultaneously induced the patients with influenza infection (29, 30) as well as the increased expression of both proinflammatory cytokines IL-2, TNF, and risk of sepsis and ARDS in chronic alcohol users and smokers, IFN-g,andanti-inflammatory IL-10 in enterotoxin A–specificVb3+ respectively (13, 14). Thus, dysfunction or depletion of AMs may be T cells (26). Thus, depletion of AMs was correlated with an overall a critical risk factor for the development of S. aureus enterotoxin– enhancement of the enterotoxin A–induced immune response. induced inflammatory diseases. Finally, CD169 is expressed not only on AMs but also other macrophages, including marginal zone macrophages in the spleen DISCLOSURES and subcapsular sinus macrophages in the lymph nodes (55). In another study, such non-AM CD169+ macrophages have been shown fi fl to capture exosomes and CD169 knockout mice had an enhanced The authors have no nancial con icts of interest. response to Ag-pulsed exosomes (56). Because enterotoxin A has been shown to permeate systemically after intranasal injection ACKNOWLEDGMENTS (26), depletion of macrophage subsets other than AMs could also contribute to the enhanced inflammatory response observed in We thank Joseph M. Ryan, Dr. Adam J. Adler, and Dr. Kepeng Wang for CD169-DTR mice. Similarly, it is also possible that DT injection and insightful discussions.

https://doi.org/10.4049/immunohorizons.1700033 ImmunoHorizons CD169+ CELLS ATTENUATE S. AUREUS ENTEROTOXIN RESPONSE 221

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