Probiotic Exopolysaccharide Protects against SystemicStaphylococcus aureus Infection, Inducing Dual-FunctioningMacrophages That Restrict Bacterial Growth and LimitInflammation

Wonbeom Paik,a Francis Alonzo III,a Katherine L. Knighta

aDepartment of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois, USA

ABSTRACT Staphylococcus aureus causes severe systemic infection with high mor-tality rates. We previously identified exopolysaccharide (EPS) from a probiotic, Bacil-lus subtilis, that induces anti-inflammatory macrophages with an M2 phenotype andprotects mice from Citrobacter rodentium-induced colitis. We tested if EPS could pro-tect from systemic infection induced by S. aureus and found that EPS-treated micehad enhanced survival as well as reduced weight loss, systemic inflammation, andbacterial burden. While macrophages from EPS-treated mice display an M2 pheno-type, they also restrict growth of internalized S. aureus through reactive oxygen spe-cies (ROS), reminiscent of proinflammatory phagocytes. These EPS-induced macro-phages also limit T cell activation by S. aureus superantigens, and EPS abrogatessystemic induction of gamma interferon after infection. We conclude that B. subtilisEPS is an immunomodulatory agent that induces hybrid macrophages that bolsterantibacterial immunity and simultaneously limit inflammation, reducing disease bur-den and promoting host survival.

KEYWORDS inflammation, macrophages, probiotics, S. aureus

Staphylococcus aureus is a Gram-positive bacterium that causes a wide array ofhuman diseases (1). S. aureus is generally found on skin and in anterior nares with

no apparent harm (2), but upon entering the bloodstream, it can disseminate andcolonize virtually all organs, resulting in severe systemic disease with up to 50%mortality (1, 3). The host rapidly recognizes the pathogen upon invasion and mountsproinflammatory responses targeted to clear the infection. However, S. aureus evadesand resists many aspects of host immunity (4), resulting in devastating outcomesmediated by overt inflammation and tissue damage. Antibiotics that directly target S.aureus are the only option for patients with systemic S. aureus disease, but widespreadprevalence of antibiotic-resistant strains limits treatment efficacy and many patientsexperience persistent bacteremia or succumb to disease despite aggressive therapy (1,5, 6). Novel approaches for treatment and prevention of systemic S. aureus infectionsare critically needed.

Within tissues, S. aureus is rapidly recognized by host pattern recognition receptors,and polymorphonuclear cells (PMN) with potent antibacterial activity are recruited (7).However, S. aureus resists antimicrobial activities of PMN, requiring additional immunecells to control infection (4, 7). Macrophages (M�) are professional phagocytes thathave antibacterial activity (8), facilitate recruitment of immune cells, coordinate adap-tive immunity, and promote resolution of inflammation and wound healing (9, 10). M�

can exhibit several functions due to their extensive heterogeneity (9). Generally, M� areclassified into two polarized states: classically activated M1 M�, associated with anti-microbial activity, and alternatively activated M2 M�, associated with immune regula-

Citation Paik W, Alonzo F, III, Knight KL. 2019.Probiotic exopolysaccharide protects againstsystemic Staphylococcus aureus infection,inducing dual-functioning macrophages thatrestrict bacterial growth and limitinflammation. Infect Immun 87:e00791-18.https://doi.org/10.1128/IAI.00791-18.

Editor Nancy E. Freitag, University of Illinois atChicago

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Katherine L.Knight, kknight@luc.edu.

Received 21 October 2018Accepted 26 October 2018

Accepted manuscript posted online 5November 2018Published

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tion and wound healing (11). M2 M� can be further characterized in four subsets (12,13), increasing overall heterogeneity of M�. This heterogeneity allows the M� com-partment to coordinate immune responses during infection, limiting bacterial growthand inflammation while promoting tissue repair to restore homeostasis. S. aureus,however, can subvert M� functions by each of several mechanisms (8, 14–16), and anattractive strategy to reduce disease burden would be to modulate M� in a mannerthat would bolster antibacterial immunity and promote resolution of inflammation.

Probiotics are microorganisms that can be used to benefit the host, and numerousprobiotic products are widely available (17). However, the specific mechanisms bywhich probiotics function remain mostly unknown. Bacillus subtilis is a Gram-positivespore-forming probiotic bacterium that can limit murine colitis induced by the entericpathogen Citrobacter rodentium (18, 19). We demonstrated that protection by B. subtilisfrom disease caused by C. rodentium requires production of exopolysaccharide (EPS)(19), and administration of purified EPS to mice via intraperitoneal (i.p.) injectionrecapitulates this protection (20). Further, we found that i.p. injection of EPS results inthe induction of peritoneal M� that display an M2 phenotype and inhibit T cellactivation in vitro and in vivo (21), suggesting that EPS mediates its protection byinducing anti-inflammatory M2 M�. We hypothesized that this anti-inflammatoryproperty of EPS could be used to limit inflammation during systemic S. aureus infection,preserving tissue integrity and thereby improving outcomes.

Here, we demonstrate that mice treated with EPS exhibit reduced weight loss,systemic inflammation, and bacterial load during S. aureus bloodstream infection,leading to increased survival. Mechanistically, we found that EPS abrogates earlyinduction of gamma interferon (IFN-�) in vivo during infection and that M� fromEPS-treated mice restrict growth of internalized S. aureus ex vivo through enhancedproduction of reactive oxygen species (ROS). Further, these M� retained their capacityto suppress T cell activation induced by S. aureus superantigen (SAg)-containing culturemedium. These data suggest that EPS induces cells possessing both antibacterial andanti-inflammatory properties, thereby providing the means to bolster S. aureus clear-ance and also maintain tissue integrity, processes that would improve the outcomes oflethal S. aureus infection.

RESULTSAttenuation of S. aureus bloodstream infection by B. subtilis-derived EPS. EPS

prevents systemic inflammation by inducing M2 M� that suppress T cell activation (21),and we hypothesized that EPS could also improve the outcome of systemic infection byS. aureus, where inflammation plays a role in pathogenesis (4, 22). We administered EPS1 day prior to and 1 day after systemic infection with an epidemic strain of S. aureusUSA300 (LAC) and found that EPS-treated mice lost less weight than phosphate-buffered saline (PBS)-treated infected controls (Fig. 1A). Even a single dose of EPSbenefitted the host, since we observed reduction in weight loss by 1 day postinfection(dpi), prior to the second administration of EPS (Fig. 1A). EPS-treated mice also hadimproved survival compared to that of PBS-treated mice (Fig. 1B). Given that EPS limitsinflammation in disease caused by infection with C. rodentium (20, 21) and reducesweight loss early in systemic S. aureus infection, we assessed if the levels of serumproinflammatory chemokines and cytokines were decreased in EPS-treated mice. In-deed, these mice had lower levels of MCP-1 (CCL2), MIP-1� (CCL3), MIP-1� (CCL4),tumor necrosis factor (TNF), and gamma interferon (IFN-�) than did PBS-treated mice at1 dpi (Fig. 1C), indicating that EPS limits systemic inflammation early during S. aureusinfection. While EPS does not appear to affect pathogen colonization in C. rodentium-induced colitis and anti-inflammatory M2 M� are not generally associated with en-hanced antibacterial immunity (19, 23, 24), we nevertheless assessed bacterial loadwithin organs of S. aureus-infected mice. We did not observe any difference in S. aureusCFU in the kidneys from EPS-treated mice at either 1 or 3 dpi (Fig. 2A). However, thebacterial burden in these mice was reduced 3-fold in the blood 6 h postinfection (hpi)(Fig. 2B). In addition, the number of S. aureus CFU was reduced in the spleen and the

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liver 8-fold and 3-fold, respectively, 1 dpi (Fig. 2C and D) and by 4-fold in the liver 3 dpi(Fig. 2D). These data indicate that EPS limits bacterial burden during systemic S. aureusinfection.

ROS-mediated inhibition of S. aureus growth by EPS-induced M�. With acute S.aureus infection, monocytes are recruited to sites of infection, where they differentiateinto classically activated, proinflammatory M1 M� that mediate antibacterial immunityand control bacterial load (8, 9). On the contrary, M2 M� generally have reducedantimicrobial functions, such as nitric oxide production and secretion of proinflamma-tory cytokines, and are thought to enhance bacteremia and sepsis (23, 24). How, then,could EPS treatment, which induces M2 M�, lead to reduced bacterial load? Wehypothesized that EPS could increase the uptake of S. aureus by immune cells and/orenhance their bactericidal activity, leading to reduced numbers of bacteria duringinfection. To test this, we isolated M� (F4/80�) from the peritoneal cavity of EPS-treated mice and incubated them with serum-opsonized S. aureus. After washing thecells free of bacteria, we determined S. aureus CFU in cell lysates. No significantdifference in the number of internalized S. aureus CFU between cells from PBS- or

FIG 1 Effect of B. subtilis EPS on disease after S. aureus bloodstream infection. Mice were treated with EPS (1 to 3 mg/kg) or PBS 1 dayprior to and 1 day after systemic infection with S. aureus. (A) Body weight loss. Differences were analyzed using two-way analysis ofvariance (ANOVA) with Bonferroni’s multiple-comparison test. Error bars represent standard deviations (SD). (B) Percent survival asdetermined by �20% body weight loss. Differences in curves were analyzed using log-rank (Mantel-Cox) test. n � 17 (PBS) or n � 22 (EPS).Data were pooled from 7 independent experiments. (C) Serum level (1 dpi) of cytokines and chemokines measured by CBA. n � 12 to 16.Data were pooled from 3 to 4 independent experiments. Each dot represents data from individual mice. Bar represents means. Data wereanalyzed using unpaired, two-tailed Student’s t test. IFN-� data were analyzed using one-sample t test. *, P � 0.05; **, P � 0.01; ***,P � 0.001.

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EPS-treated mice was found (Fig. 3A, 0.5 h, left). We continued to culture the infectedcells and found that by 5 h, the numbers of S. aureus CFU were significantly lower incultures of cells from EPS-treated mice (Fig. 3A, left), indicating that cells from thesemice restrict growth of internalized S. aureus more than cells from PBS-treated mice.EPS did not directly suppress S. aureus growth, since its growth in vitro was unaffectedby the presence of EPS (up to 0.5 mg/ml) in culture medium (see Fig. S1 in thesupplemental material). Further, F4/80� cells from EPS-treated mice did not limitgrowth of S. aureus (Fig. 3A, right), indicating that EPS exerts its effect specificallythrough M�.

Phagocytes utilize multiple mechanisms to respond to internalized pathogens (8),but one common mechanism is through production of ROS that induce oxidativedamage and kill bacteria (8, 25). After uptake, M� generate ROS through an NADPHoxidase complex that catalyzes the reaction between O2 and NADPH to generate O2

�,which is used to generate a variety of reactive oxidants, a process known as respiratoryburst (25). Since EPS-induced M� inhibit growth of internalized S. aureus, we thought

FIG 2 Effect of EPS on S. aureus burden during bloodstream infection. Mice were treated with EPS or PBS1 day prior to systemic infection with S. aureus, and bacterial CFU in different organs were determined.(A) Numbers of S. aureus CFU per organ in kidney 1 and 3 dpi. (B) Numbers of S. aureus CFU/ml in blood6 hpi. (C) Numbers of S. aureus CFU per organ in spleen 1 dpi. (D) Numbers of S. aureus CFU per organin liver 1 and 3 dpi. n � 8 to 10 (6 hpi or 1 dpi) or n � 17 to 18 (3 dpi). Data were pooled from 2 to 5independent experiments. Each dot represents an individual mouse. Bars represent means. Data wereanalyzed using unpaired, two-tailed Student’s t test. *, P � 0.05; **, P � 0.01; ***, P � 0.001.

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that EPS may increase ROS levels in M� during infection with S. aureus, therebyenhancing their antibacterial capacities. We isolated total peritoneal cells from EPS-treated mice and infected them with serum-opsonized S. aureus. After washing infectedcells, we measured the level of ROS in CD11bhigh F4/80high M� by flow cytometry usingan ROS indicator, CellROX green. Although we did not find differences in levels ofCellROX staining between uninfected M� isolated from PBS- or EPS-treated mice (Fig.3B), upon infection, the M� from EPS-treated mice had increased CellROX greenstaining while those from PBS-treated mice did not (Fig. 3B), indicating that M� fromEPS-treated mice increase cellular ROS levels in response to S. aureus infection. Wehypothesized that this increase in ROS levels is responsible for inhibition of S. aureusgrowth by M� from EPS-treated mice and tested this by repeating the assay shown in

FIG 3 Effect of EPS on M� capacity to inhibit growth of internalized S. aureus. Mice were treated withEPS or PBS, and 3 days later peritoneal cells were isolated. (A) Peritoneal cells were sorted into F4/80�

and F4/80� cells using magnetic selection and infected with S. aureus for 30 min; CFU were monitoredover time. Differences were analyzed using two-way ANOVA with Bonferroni’s multiple-comparison test.n � 5 to 8. Data were pooled from 3 to 4 independent experiments. (B) Peritoneal cells were infected withS. aureus and stained with CellROX green. Cells were analyzed by flow cytometry after gating on CD11bhigh

F4/80high cells. Data were normalized to uninfected cells from PBS-treated mice. Differences were analyzedusing one-way ANOVA with Bonferroni’s multiple-comparison test. n � 9. Data were pooled from 3 indepen-dent experiments. (C) Same setup as that for panel A but in the presence or absence of 2 �M DPI. Differenceswere analyzed using 2-way ANOVA with Bonferroni’s multiple-comparison test. n � 6. Data were pooled from3 independent experiments. Error bars represent SD in panels A and C, and bars represent means in panel B.Each dot represents data from a single mouse. *, P � 0.05; **, P � 0.01; ***, P � 0.001.

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Fig. 3A in the presence of diphenyleneiodonium (DPI), a noncompetitive NADPHoxidase inhibitor. We found that the inhibition of growth of internalized S. aureus byM� from EPS-treated mice was abrogated in the presence of DPI (Fig. 3C), suggestingthat these M� inhibit growth of S. aureus through ROS.

Inhibition of S. aureus superantigens by EPS-induced M�. EPS induces anti-inflammatory M2 M� that limit T cell activation (21), but M2 M� are not generallyassociated with bactericidal activity (24). Paradoxically, as shown above, we found thatEPS-induced M� inhibit S. aureus growth through ROS, like that of proinflammatory M1M� (24). M� have a broad spectrum of functions (11) and are plastic in nature,constantly modulating their functions in response to environmental factors (26). Amajor pathway by which innate immune cells are activated by S. aureus is the Toll-likereceptor 2 (TLR2)-myeloid differentiation primary response 88 (MyD88) pathway (27),which recognizes S. aureus-derived lipoproteins (28) and induces proinflammatoryactivation of M� (24). We hypothesized that upon exposure to S. aureus and itsactivating ligands, EPS-induced M� could change to a proinflammatory phenotype andgain antibacterial function. In this case, we expected that M2 M� polarization would belost and the M� would no longer inhibit T cell activation. To test this possibility, wedevised a T cell activation assay using S. aureus culture supernatants that contain bothS. aureus lipoproteins that drive proinflammatory activation of M� (8, 28) and super-antigens (SAg) that drive T cell activation and proliferation (29). The results show thatwhereas S. aureus culture supernatants of LAC and JE2 strains (both USA300 lineages),which produce staphylococcal enterotoxin-like (SEl)-K, -Q, and -X SAgs (30), stimulatedsplenic T cell proliferation, the supernatant from an SEl-Q-deficient mutant (selQ::Tn) didnot (Fig. 4 and Fig. S2), indicating that T cell activation was dependent on SEl-Q SAg.Deficiency in another gene encoding a putative type A enterotoxin (USA300_1559::Tn)did not affect T cell activation (Fig. 4 and Fig. S2). To test if EPS-induced M� inhibitSEl-Q-mediated T cell proliferation in the presence of inflammatory factors of S. aureus,we cultured peritoneal F4/80� M� from PBS- or EPS-treated mice with naive spleno-cytes that were stimulated with culture supernatant of S. aureus USA300 (LAC). Flowcytometric analysis showed a decrease in CD25� CD44�-activated CD4� and CD8� Tcells (Fig. 5A and Fig. S3) as well as reduced T cell proliferation (Fig. 5B and Fig. S3),showing that EPS-induced M� still inhibit T cell activation by S. aureus SEl-Q SAg, evenin the presence of other proinflammatory S. aureus factors.

SAgs are a classical virulence factor of S. aureus that cross-link T cell receptors (TCR)and major histocompatibility complex (MHC) molecules on accessory cells to drivepolyclonal T cell activation and proinflammatory cytokine production, resulting in toxicshock syndrome (31). Importantly, SAg induces IFN-� production early in S. aureussystemic infection, thereby promoting pathogen survival in vivo (32). Because EPS

FIG 4 Effect of S. aureus SAg on CD4� and CD8� T cell proliferation. CTV-labeled splenocytes from naivemice were cultured in the presence of 33% culture supernatant of S. aureus or SAg-deficient mutants for4 days and analyzed for T cell proliferation by flow cytometry. Proliferative index represents thepercentage of events within the proliferated gate, as shown in Fig. S2. Error bars represent SD. n � 4 to6. Data were pooled from 2 independent experiments. Data were analyzed using one-way ANOVA withBonferroni’s multiple-comparison test. ***, P � 0.001.

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abrogated levels of early IFN-� production during S. aureus infection (Fig. 1C), wehypothesized that EPS inhibits the stimulatory effects of S. aureus SAgs. We tested thisby isolating splenocytes from PBS- or EPS-treated mice and stimulating them ex vivowith S. aureus culture supernatants. Indeed, splenocytes from EPS-treated mice hadreduced T cell proliferation compared to that of cells from PBS-treated mice (Fig. 6 andFig. S4). We conclude that EPS-induced M� retain their anti-inflammatory function tolimit T cell activation by S. aureus in vitro and that splenic T cells from EPS-treated micehave a diminished response to S. aureus SAg.

Induction of hybrid M1-M2 M� by EPS. M� are generally described as proinflam-matory M1 or anti-inflammatory M2. M� have been studied primarily using in vitropolarization with lipopolysaccharide (LPS) and IFN-�, which induce classical M1 M� that

FIG 5 Effect of EPS-induced M� on CD4� and CD8� T cell activation by S. aureus SAg. CTV-labeled naivesplenocytes were cocultured with peritoneal F4/80� cells from PBS- or EPS-treated mice for 4 days in thepresence of 33% S. aureus culture supernatant and analyzed for T cell activation (A) and proliferation (B)by flow cytometry. Data were pooled from 3 independent experiments. Bars represent means. n � 8.Data were analyzed using Student’s t test. *, P � 0.05; **, P � 0.01.

FIG 6 Effect of EPS on splenic CD4� and CD8� T cell responses to S. aureus SAg. CTV-labeled splenocytesfrom PBS- or EPS-treated mice were cultured for 4 days in the presence of 33% S. aureus culturesupernatant and analyzed by flow cytometry. Data were pooled from 3 independent experiments. NT,not tested. Bars represent means. n � 6 to 7. Data were analyzed using one-way ANOVA with Bonferroni’smultiple-comparison test. *, P � 0.05; ***, P � 0.001.

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express proinflammatory cytokines and inducible nitric oxide synthase (iNOS), and withinterluekin-4 (IL-4) and IL-13, which induce alternative M2 M�, as defined by arginase-1(Arg-1) expression (11, 12). Both iNOS and Arg-1 utilize arginine as their substrate, andit is thought that these two enzymes negatively regulate each other, contributing topolarization of M� (33). However, M� are plastic in nature and hybrid M1/M2 M� havebeen reported (34). Because EPS-induced M� inhibit S. aureus growth through ROS, asdo M1 M�, and also inhibit T cell activation by S. aureus SAg, as do M2 M�, wehypothesized that EPS-induced M� represent hybrid M1- and M2-like M�. To testthis possibility, we assessed expression of iNOS and Arg-1 in peritoneal CD11bhigh

F4/80high M� in EPS-treated mice and found a significant increase in both iNOS andArg-1 expression, indicating that EPS-induced M� coexpress M1 and M2 M�

markers (Fig. 7). These data further indicate that EPS induces a hybrid M1- andM2-like M� in vivo.

DISCUSSION

Systemic infection by S. aureus is a severe challenge to the host, requiring strongantibacterial immunity while limiting overt inflammation. We demonstrated that B.subtilis-derived EPS protects mice from systemic S. aureus infection, and that thisprotection is likely due to reduction in bacterial load and limitation of inflammation byM� that both suppress S. aureus growth through ROS and limit T cell activation by SAg.EPS-treated mice display reduced serum levels of proinflammatory cytokines andchemokines within 24 h after S. aureus infection. A surprising finding was the reducedlevel of IFN-� in EPS-treated mice infected with S. aureus. Classically, IFN-� is the

FIG 7 Effect of EPS on M1 and M2 M� marker expression levels. Peritoneal CD11bhigh F4/80high M� fromPBS (solid line)-, ΔEPS (dotted line)-, or EPS (dashed line)-treated mice were analyzed for M2 (Arg-1) andM1 (iNOS) markers by flow cytometry. (A) Representative flow cytometry histogram plot. (B) Medianfluorescence intensity compared to that of PBS-treated M�. Data were pooled from three independentexperiments. n � 5 to 10. Data were analyzed using one-way ANOVA with Bonferroni’s multiple-comparison test. **, P � 0.01; ***, P � 0.001.

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defining agent of type 1 immunity (35) and mediates protection against bacterialinfections by enhancing antimicrobial functions of phagocytes (36–38). Although ad-ministration of recombinant IFN-� led to improved survival in murine systemic S. aureusinfection in one study (39), other studies showed that neutralization of IFN-� or IFN-�deficiency improved survival of S. aureus-infected mice with reduced bacterial burdenin the kidney (40, 41), suggesting that IFN-� accentuates disease. Our data are consis-tent with the idea that IFN-� contributes to S. aureus pathogenesis, because diseasewas attenuated in EPS-treated mice that had decreased levels of IFN-�.

IFN-� is produced primarily by CD4� T cells (29, 37, 42), and SAgs are major S. aureusvirulence factors that induce T cells as well as NKT cells to produce IFN-� (29, 43).Whereas infection with wild-type S. aureus increases serum IFN-� levels within 8 h,infection with a Δsea mutant (deficient in staphylococcal enterotoxin A SAg) does not,and bacterial load is reduced in the liver by 4 dpi (32). These findings suggest that SAgdrives the production of IFN-�, which promotes bacterial survival, although the patho-genic process for this is not known. Our finding that EPS-induced M� inhibit S. aureusSAg activation of splenic T cells suggests that EPS functions in vivo by limiting SAgactivation of T cells and subsequent IFN-� production, thereby neutralizing the patho-genic effects of IFN-�. The precise role of IFN-�, a cytokine classically associated withenhanced antibacterial immunity, plays in S. aureus pathogenesis provides an interest-ing avenue of study to better understand how pathogens exploit host immunity topromote its survival and disease.

The mechanism by which EPS-induced M� inhibit S. aureus-induced T cell activationis not known. Although it could result from decreased expression of MHC class IImolecules, which are recognized by SAgs, EPS does not alter MHC class II expression onM� (unpublished data). Instead, because we previously showed that EPS-induced M�

inhibit anti-CD3� activation of T cells through PD-L1 and transforming growth factorbeta (TGF-�) and that EPS promotes regulatory T (Treg) cells, we suggest that theinhibition of T cell activation by S. aureus is due to the upregulated expression ofinhibitory molecules such as PD-L1, PD-L2, TGF-�, or IL-10 or to the generation ofTreg cells (21). Alternatively, the inhibition may be due to depletion of argininethrough iNOS and Arg-1 (44–46), both of which are expressed in EPS-induced M�.

EPS reduces bacterial burden during S. aureus infection in blood, spleen, and liverbut not in the kidney. We suggest that the difference in S. aureus CFU is due todifferences in the response to SAg stimulation. Infection with a SAg-defective mutantresulted in reduced numbers of S. aureus CFU in the liver but not in kidney (32),indicating that SAg promotes colonization in the liver but not kidney. Kidney, com-pared to liver and spleen, contains relatively few T and NKT cells, targets of SAg.Because EPS inhibits SAg stimulation of T cells, we think the effect of EPS is primarilyin the liver and spleen, where large numbers of T and NKT cells reside (29, 43). Anotherexplanation for the difference in the number of bacterial CFU between organs maycome from the myeloid compartment. In the kidney, dendritic cells (DC) serve assentinels for infection and recruit neutrophils (47), which leads to abscess formation. Incontrast, liver harbors Kupffer cells, unique tissue resident M�, that clear activatedneutrophils and cellular debris (48, 49). Clearance of neutrophils would reduce abscessformation within hepatic tissues and not only help clear bacteria but also maintain M�

access to S. aureus. In addition, peritoneal M� are known to traffic directly to the liverduring liver injury to clear activated neutrophils and promote wound repair (50),providing another means of reducing pathogenic neutrophil activation and supple-menting M�-mediated immunity against S. aureus. The spleen also contains specializedresident M� subsets known to promote immunity against bloodborne pathogens (51),and they could contribute to clearing activated neutrophils and provide additionalimmunity against S. aureus.

EPS induces macrophages that coexpress M1 and M2 markers and display functionsof both M1 and M2 macrophages. Subdivision of macrophages into M1 or M2 is basedon in vitro polarization conditions, and in vivo, the functions of macrophages span awide spectrum (11, 12, 26). For example, Arg-1� myeloid cells (M2-like) are associated

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with persistent S. aureus biofilms (23), but they also inhibit growth of planktonic S.aureus during infection (M1-like) (52). Because of this, we think that EPS-induced M�

display both M1- and M2-like properties due to as-yet-unidentified environmental cuesgenerated during EPS treatment in vivo. While we cannot rule out the possibility thatEPS-induced M� contain two distinct groups, one M1-like and one M2-like, hybridstates of M� have been described (34), and by flow cytometry, it appears thatessentially all M� upregulate expression of M1 and M2 markers in response to EPS.

Probiotics are widely marketed for their health benefits (17, 53), but the specificmechanisms by which these products benefit the host are mostly unknown (54). Further,the molecules and their mechanism of action are poorly understood (20, 55–59), with a fewexceptions, including polysaccharide A (PSA) and �-galactosylceramide from Bacteroidesfragilis (60–62). PSA is a zwitterionic polysaccharide that modulates T cell responses throughDCs in a TLR2-dependent manner (61) and protects mice from experimental colitis throughan IL-10-dependent mechanism (63). �-Galactosylceramide functions through CD1d-mediated recognition by NKT cells (62). While the structure of B. subtilis EPS is not yetknown, it harbors immunomodulatory properties different from those of other knownprobiotics, because the induction of anti-inflammatory responses is TLR4 dependent andthe inhibition of T cell responses is not through IL-10 but through TGF-� and PD-L1 (21).Further, these M� not only limit inflammation but also promote antibacterial immunityagainst an invasive pathogen. Additional knowledge of the structural and functionalproperties of EPS is needed to understand and appreciate its full potential as an immuno-modulatory molecule.

In conclusion, B. subtilis EPS represents a recently discovered probiotic-derivedagent that protects hosts from systemic S. aureus infection by bolstering antibacterialimmunity of phagocytes through enhanced ROS production and by limiting inflamma-tory activation of immune cells. We suggest that this multifunctionality allows EPS tocounteract several aspects of S. aureus pathogenesis to improve outcomes, an attrac-tive strategy to combat complex systemic infections.

MATERIALS AND METHODSMice and reagents. C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME) and

bred in-house at Loyola University Chicago. Four- to 8-week-old mice were used for all studies. We didnot observe any noticeable differences between male and female mice. All experiments were performedaccording to protocols approved by the Institutional Animal Care and Use Committee at LoyolaUniversity Chicago. Cell culture base medium and supplements were from Life Technologies (GrandIsland, NY), bacterial media were from BD (Franklin Lakes, NJ), and antibodies were from BioLegend (SanDiego, CA). Starter cultures of S. aureus USA300 strain LAC or its plasmid-cured derivative, AH1263 (64),were prepared in tryptic soy broth (TSB). These two strains were used interchangeably, and we did notobserve notable differences in outcomes. Transposon insertion mutants (sel-k::Tn, sel-q::Tn, sel-x::Tn, andUSA300_1559::Tn) from the University of Nebraska transposon mutant library were used for SAg studies(65). For bloodstream infection experiments, overnight cultures were diluted 1:100 into fresh growthmedium, incubated at 37°C to exponential phase (�3 h), and subsequently normalized to an inoculumof 108 CFU/ml in PBS on the day of infection.

Preparation of B. subtilis-derived EPS. EPS was prepared as previously described (17, 18), with afew modifications. B. subtilis DS991 (sinR::kan tasA::spec; overproduces and secretes EPS) or DK4623 (ΔEPS;sinR::kan tasA::spec sdpABC::mls skf::tet lytC::cat ΔPBSX ΔSPB ΔpBS32, DS991 with lytic genes [66] andprophages deleted), generously provided by Daniel B. Kearns (Indiana University, Bloomington, IN), wascultured in 1% tryptone-phosphate broth (1% tryptone, 25 mM phosphate, 0.1 M NaCl) or Msgg (minimalsalts glutamate glycerol) medium. EPS was obtained from stationary-phase culture supernatants (21) orfrom bacterial lawns on Luria-Bertani agar plates by 75% ethanol precipitation at �20°C. The precipitatewas pelleted by centrifugation (13,700 g, 4°C, 30 min), resuspended in 0.1 M Tris (pH 8), and treatedwith DNase (67 �g/ml) and RNase (330 �g/ml) at 37°C for 2 h, followed by proteinase K (40 �g/ml)digestion at 55°C for 2 h. EPS was then purified by DEAE-cellulose (Whatman, Maidstone, UK) ionexchange chromatography and/or gel filtration on Sephacryl S-500. Carbohydrate-positive fractions wereidentified by a modified phenol sulfuric acid assay (67, 68) and desalted by gel filtration (Pharmacia FineChemicals, Piscataway, NJ). Total carbohydrate content was measured using the modified phenol sulfuricacid assay. All EPS preparations were assessed for protein and nucleic acid content by spectrometry andalso for their ability to induce peritoneal M2 M�, as previously described (21), prior to use.

Murine model of S. aureus bloodstream infection. C57BL/6J mice were treated with 3 mg/kg ofbody weight EPS by intraperitoneal (i.p.) injection in a 200-�l final volume in PBS 1 day before and 1 dayafter infection. Control mice were injected with equal volumes of PBS. On day 0, anesthetized mice wereinfected with 107 CFU S. aureus in 100 �l PBS by retro-orbital injection (69, 70). Mice with �20% bodyweight loss that showed signs of lethargy were euthanized during the experiment. While most mice were

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assessed until 3 dpi, any mice that lost �20% body weight were considered dead for assessing survivalin order to avoid utilizing subjective criteria for moribund status. For disease assessment, numbers of S.aureus CFU in spleen, liver, and kidney homogenates of euthanized mice were determined by plating ontryptic soy agar plates for 12 h at 37°C. Some of the surviving EPS-treated mice were randomlyselected (4 mice from 2 independent experiments) and were monitored for 13 days for long-termsurvival analysis.

Serum cytokine measurements. Serum was collected from mice 1 dpi, and levels of proinflamma-tory cytokines and chemokines were measured using a cytometric bead array (CBA) (BD Biosciences)according to the manufacturer’s specifications. Samples were analyzed on an LSRFortessa (BD Biosci-ences), and data were analyzed using FlowJo software (Ashland, OR) by gating on individual beads andexamining the geometric mean of detection reagent fluorescence intensity.

S. aureus uptake and growth with peritoneal phagocytes. Three days after EPS injection, perito-neal cells were harvested by lavage with RPMI medium. F4/80� macrophages were purified using the BDIMag cell separation system (BD Biosciences) after incubation with anti-CD16/32 (mouse BD Fc block,clone 93) and biotinylated anti-F4/80 (BM8) monoclonal antibodies. F4/80� and F4/80� cells (5 105) ortotal peritoneal cells (106) were infected with S. aureus and opsonized by incubation with 10% mouseserum for 30 min at 37°C, at multiplicity of infection of 1, for 30 min in antibiotic-free RPMI. S. aureusuptake was assessed by washing cells three times, lysing cells with 0.1% saponin, and plating on solidmedium to quantify S. aureus CFU. S. aureus growth was assessed after 2 to 6 h in culture, followed byquantifying S. aureus CFU after lysis with 0.1% saponin and plating on solid medium. For ROS experi-ments, cells were washed of free bacterium and then further incubated in medium containing 1.25 �MCellROX green (Thermo Fischer Scientific, Waltham, MA) for 1 h in rotating tubes. Cells were thenincubated with anti-CD16/32 (93), stained with anti-CD11b-allophycocyanin (APC) (M1/70) and anti-F4/80-APC-Cy7 (BM8) antibodies, and analyzed using an LSRFortessa (BD Biosciences). Flow cytometric datawere analyzed using FlowJo software by gating first for nonlymphocytes and then for CD11bhigh

F4/80high M�, followed by assessing median fluorescence intensity of CellROX green staining. Data werenormalized to those for uninfected cells from PBS-treated mice for each experiment. For assessing theeffect of ROS on bacterial growth, 2 �M diphenyleneiodonium (DPI; Sigma-Aldrich, St. Louis, MO) wasincluded in the medium.

S. aureus SAg-mediated activation of splenocytes. Splenocytes from PBS- or EPS-treated C57BL/6Jmice were labeled with CellTrace violet (CTV; Life Technologies) according to the manufacturer’sdescriptions, and 3.5 105 cells were cultured in 96-well flat-bottom plates (Corning, Corning, NY). Cellswere stimulated with cell-free supernatants of stationary-phase cultures of S. aureus AH1263 or JE2transposon library mutants in RPMI medium at a final dilution of 1:3. After 4 days, nonadherent cells werestained with anti-CD4-APC (GK1.5), anti-CD8a-PE-Cy7 (53-6.7), anti-CD25-APC-Cy7 (PC61), and anti-CD44-PE (IM7) antibodies. Cells were analyzed using an LSRFortessa or FACSCanto II, and data wereanalyzed using FlowJo software by gating on CD4� and CD8a� lymphocytes. For coculture studies,peritoneal F4/80� M� (2.5 104) were obtained from PBS- or EPS-treated mice using the BD IMag cellseparation system (BD Biosciences).

Flow cytometric analysis of M1 and M2 markers. Peritoneal cells from PBS- or EPS-treatedC57BL/6J mice were incubated with anti-CD16/32 (93) and stained with anti-CD11b-APC (M1/70) andanti-F4/80-APC-Cy7 (BM8) antibodies. Cells were then fixed and permeabilized (BD Cytofix/Cytoperm) forintracellular staining with anti-Arg-1-fluorescein isothiocyanate (R&D Systems) and anti-iNOS-phycoerythrin-eFluor610 (BD Pharmingen) antibodies. Cells were analyzed using LSRFortessa, and data were analyzed usingFlowJo software by gating on CD11bhigh F4/80high M�.

Statistical analyses. Statistical significance was determined by an unpaired, two-tailed Student’s ttest, unless otherwise noted, using GraphPad Prism software (La Jolla, CA).

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at https://doi.org/10.1128/IAI

.00791-18.SUPPLEMENTAL FILE 1, PDF file, 0.1 MB.SUPPLEMENTAL FILE 2, PDF file, 0.2 MB.SUPPLEMENTAL FILE 3, PDF file, 0.2 MB.SUPPLEMENTAL FILE 4, PDF file, 0.1 MB.

ACKNOWLEDGMENTSWe thank Daniel B. Kearns (Indiana University) for generously providing the B.

subtilis strains used in this study and Mae Kingzette for purification of EPS.

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