Impact of Staphylococcus Aureus USA300 Colonization and Skin Infections on Systemic Immune Responses in Humans

Impact of Staphylococcus aureus USA300 Colonization and Skin Infections on Systemic Immune Responses in Humans

This information is current as Maria-Luisa Alegre, Luqiu Chen, Michael Z. David, of September 24, 2021. Caroline Bartman, Susan Boyle-Vavra, Neha Kumar, Anita S. Chong and Robert S. Daum J Immunol 2016; 197:1118-1126; Prepublished online 11 July 2016;

doi: 10.4049/jimmunol.1600549 Downloaded from http://www.jimmunol.org/content/197/4/1118

Supplementary http://www.jimmunol.org/content/suppl/2016/07/11/jimmunol.160054 Material 9.DCSupplemental http://www.jimmunol.org/ References This article cites 39 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/197/4/1118.full#ref-list-1

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Impact of Staphylococcus aureus USA300 Colonization and Skin Infections on Systemic Immune Responses in Humans

Maria-Luisa Alegre,*,1 Luqiu Chen,*,1 Michael Z. David,*,† Caroline Bartman,‡ Susan Boyle-Vavra,† Neha Kumar,† Anita S. Chong,x and Robert S. Daum†,x

Staphylococcus aureus is both a commensal and a pathogen, and USA300, a strain that is usually methicillin-resistant but can sometimes be methicillin-susceptible, has been causing skin and soft tissue infections (SSTIs) in epidemic proportions among otherwise healthy individuals. Although many people are colonized with S. aureus strains, including some with USA300, few of these colonized individuals develop SSTIs. This prompts the hypothesis that infections may develop in individuals with somewhat reduced innate and/or adaptive immune responses to S. aureus, either because prior S. aureus colonization has dampened such responses selectively, or because of more globally reduced immune reactivity. In this study, we analyzed the S. aureus colonization status and PBMC responses to innate and adaptive stimuli in 72 patients with SSTIs and 143 uninfected demographically matched Downloaded from controls. Contrary to the hypothesis formulated, PBMCs from infected patients obtained at the time of infection displayed enhanced innate cytokine production upon restimulation compared with PBMCs from controls, a difference that disappeared after infection resolution. Notably, PBMCs from patients infected with a documented USA300 SSTI displayed greater innate cytokine production than did those from patients infected with documented non-USA300 genotypes. Moreover, colonization with USA300 in infected patients, regardless of their infecting strain, correlated with increased production of IL-10, IL-17A, and IL-22 compared with patients colonized with non-USA300 subtypes. Thus, our results demonstrate that infected patients associated with http://www.jimmunol.org/ USA300 either as an infecting strain, or as a colonizing strain, have systemic immune responses of greater magnitude than do those associated with other S. aureus subtypes. The Journal of Immunology, 2016, 197: 1118–1126.

he Gram-positive bacterium S. aureus is a frequent com- the community, although methicillin-susceptible S. aureus (MSSA) mensal of the human skin and mucosal areas, but it is also strains sharing many genetic characteristics of USA300 also exist T the most commonly isolated human bacterial pathogen (2, 3). and an important cause of skin and soft tissue infections (SSTIs) It is often cited that 20% of the human population is persistently

(1). Since the mid-1990s, there has been a sharp increase in the colonized with S. aureus (4), although the prevalence is likely by guest on September 24, 2021 incidence of S. aureus SSTIs in community-dwelling populations higher (5). Aside from patients with immune genetic defects known in the absence of frequent exposure to the healthcare system. This to be associated with recurrent S. aureus infections (6), it is not clear increase has coincided with the identification of new, virulent why some seemingly healthy individuals develop S. aureus S. aureus strain types that are usually resistant to nearly all b-lactam SSTIs and others do not. One possibility is that individuals antibiotics (methicillin-resistant S. aureus [MRSA]). Such community- with slightly lower immune responses (toward the left of the associated MRSA strains can be distinguished molecularly from Gaussian distribution of normal immune responses) are the ones health care–associated strains and appear to cause infections in who develop SSTIs. Indeed, polymorphisms in the cytokine genes otherwise healthy individuals. Since 2001, in the United States, IL6, TNF, IL10, IL17A,andIFNG, as well as in the innate immune USA300 MRSA has been the dominant strain causing infections in modulating gene TLR10, have been linked to susceptibility to complicated SSTIs, although how these polymorphisms affect cytokine levels has not been resolved (7, 8). An alternative possibility is † *Department of Medicine, University of Chicago, Chicago, IL 60637; Department of that colonization with S. aureus may tolerize the immune system to Pediatrics, University of Chicago, Chicago, IL 60637; ‡University of Pennsylvania, Philadelphia, PA 19104; and xDepartment of Surgery, University of Chicago, Chicago, S. aureus Ags or microbial products, as such predisposing an indi- IL 60637 vidual to subsequent infections by any S. aureus strain. This is 1M.-L.A. and L.C. contributed equally to this work. conceivable, as the superantigen properties of S. aureus can result in ORCIDs: 0000-0001-5707-6194 (M.-L.A.); 0000-0001-8537-6071 (L.C.); 0000- deletion or anergy of an entire Vb family of T cells (9), and de- 0002-1926-7800 (M.Z.D.). sensitization of pattern-recognition receptors can occur in innate cells Received for publication March 29, 2016. Accepted for publication June 13, 2016. following exposure to certain microbial products (10). This work was supported by National Institutes of Health Grant AR059414 (to R.S.D. S. aureus infections can activate both innate and adaptive and M.-L.A.). immune cells. The cell surface TLR2 and the cytosolic nucleotide- Address correspondence and reprint requests to Dr. Maria-Luisa Alegre, University binding oligodimerization domain 2 are the main microbial mo- of Chicago, 924 E. 57th Street, JFK R-312, Chicago, IL 60637. E-mail address: [email protected] lecular pattern-recognition receptors known to signal immune The online version of this article contains supplemental material. cells during S. aureus infection (11). Activated innate cells pro- Abbreviations used in this article: D0, day 0; D40, day 40; ED, emergency depart- duce inflammatory cytokines, including IL-6, that promote pro- ment; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin- duction of C-reactive protein, a primitive opsonin and an activator sensitive Staphylococcus aureus; PVL, Panton–Valentine leukocidin; SSTI, skin of complement (12). An association between the presence of and soft tissue infection. neutralizing anti-IL-6 Abs in the serum, low C-reactive protein, Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 and recurrent S. aureus infection has been reported (13), empha- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600549 The Journal of Immunology 1119 sizing the importance of this cytokine in anti–S. aureus immunity. of .38.5˚C), receipt of immunosuppressive medications, presence of a T cell responses have received less attention, but emerging data comorbid condition such as diabetes or chronic renal failure, morbid . 2 support their role in protection against S. aureus infections. For obesity (body mass index of 40 kg/m ), or antibacterial therapy with anti-staphylococcal activity within the prior 14 d. All inclusion/exclusion instance, T cells, but not Abs, were shown to mediate protection criteria were as described previously (27, 28). Patients were randomized to against S. aureus lethality, following vaccination in mice (14, 15). receive either trimethoprim/sulfamethoxazole or clindamycin (or a placebo A link between T cell IL-17 and IL-22 and protection against in cases of a single abscess ,5 cm in diameter). Surgical incision and SSTIs caused by S. aureus has emerged both in rodents and hu- drainage were performed when an abscess was present, and purulent material was cultured. Blood was drawn on the day of the infected patients’ mans (16–19), and our group has shown that Th17 cells can confer first visit (day 0, D0) and at day 40 (D40) after enrollment. partial protection that limits the size of dermonecrotic lesions Uninfected controls enrolled were adults presenting to the ED during the upon secondary skin infection in mice (20). Notably, patients with same time period with minor, noninfectious complaints without comor- mutations in the STAT3 gene that signals downstream of IL-6 bidities. Controls included individuals of the same age, gender, and self- develop recurrent S. aureus infections of the skin and lung, but reported race as the patients with an SSTI. Blood was drawn only when the control patient was identified (D0). not systemic infections (21–26). Because IL-6 is important for At enrollment of SSTI patients and controls, informed consent was obtained. Th17 differentiation, these patients have an absence of S. aureus– Each consenting subject was cultured at nasal, oropharyngeal, and perianal/ mediated IL-17 production by T cells. These observations suggest inguinal sites using separate dry rayon-tipped applicators (CultureSwab, BD that reduced systemic Th17 responses may result in increased Diagnostic Systems). These cultures were repeated in infected patients at the follow-up visit on D40. The protocol was approved by the Institutional Review susceptibility to local SSTIs. By extension to otherwise healthy Board of The University of Chicago. individuals, these studies suggest that evaluating systemic immunity to S. aureus by analyzing PBMCs may reveal some causes Colonization swab culture conditions and molecular diagnosis Downloaded from of susceptibility to localized skin infections. of colonization and infection isolates In this study, we set out to address specific questions related to the Culture swabs were transported to the University of Chicago MRSA Re- susceptibility to S. aureus SSTIs: 1) Do people who develop SSTIs search Laboratory and were incubated in trypticase soy broth with 7% have weaker than average immune responses? 2) Does colonization sodium chloride overnight at 37˚C. An aliquot of the broth was then spread with S. aureus result in dampened innate or adaptive reactivity to onto BBL CHROMagar S. aureus media (BD Diagnostic Systems) and incubated for 24 h at 37˚C. One mauve colony was picked from the plate S. aureus restimulation? 3) Are immune responses to a USA300 and confirmed to be S. aureus by typical colony morphology, positive http://www.jimmunol.org/ infection of different magnitude or quality than immune responses catalase, and agglutination with the Staphaureux test (Remel, Thermo to other S. aureus genotypes, explaining the increase in this specific Scientific). S. aureus isolates were stored at 270˚C in skim milk (Difco) infection? To address these questions, we took advantage of an until further evaluation. ongoing multicenter clinical trial comparing different treatment Genomic DNA was extracted from each isolate using the Qiagen DNeasy blood and tissue kit following the manufacturer’s instructions and modalities in patients presenting to the emergency department (ED) modified by incubation with lysostaphin in resuspension buffer (at 37˚C for with an SSTI, in the absence of comorbidities (27, 28). Blood was at least 30 min) to facilitate S. aureus lysis (29). S. aureus speciation was collected from patients with skin infections, and from control in- confirmed using a PCR assay specific for spa (encoding protein A). dividuals presenting to the ED for minor noninfectious causes, and S. aureus isolates were characterized by multilocus sequence typing (30) to

determine the genetic background and by typing of the SCCmec element, by guest on September 24, 2021 production of cytokines was compared in both serum and stimulated the mobile genetic element that carries mecA (31) with type assignments PBMCs. In parallel, skin swabs from three anatomic sites in SSTI using published guidelines (32). Detection of the genes encoding the patients (at sites distinct from the infection) and uninfected controls Panton–Valentine leukocidin (PVL) was performed as described (33). Two were cultured to identify S. aureus colonization. The infecting SSTI S. aureus isolates were considered indistinguishable when they shared a S. aureus isolate also underwent genotypic characterization. Our multilocus sequence typing and SCCmec type and were concordant with respect to the presence or absence of the PVL genetic determinants. Based results document the systemic immune response that takes place on a previous investigation demonstrating that the presence of both the following SSTIs, reveal stronger immune responses to USA300 PVL genes (luk-S-PV and luk-F-PV) and the arcA gene found on ACME than non-USA300 strain types, and argue against systemic immu- is highly concordant with USA300 MRSA genetic background assessed nosuppressive effects by colonizing S. aureus strains. by pulsed ﬁeld gel electrophoresis (34), isolates with these characteristics were considered to be USA300 MRSA (Table I). USA300 isolates that lacked mecA and an SCCmec element were termed USA300 MSSA. Materials and Methods Patients and samples Preparation and isolation of serum, PBMCs, and cytokine assays The patients enrolled were otherwise healthy adults who presented to the ED at the University of Chicago Department of Medicine with a skin in- Serum was prepared from ∼3 ml of nonheparinized blood by centrifuging the fection between September 2010 and September 2012. Exclusion criteria tube at 3000 rpm for 10 min at room temperature. The serum was collected included a human or animal bite at the infection site, fever (oral temperature and aliquots were frozen at 220˚C. Cytokine levels of IL12p70, TNF, IL-1b,

Table I. Algorithm for classiﬁcation of S. aureus strains

Strain Type mecA PVL arcA ST SCCmec Type USA300 MRSA USA300 + + + Not done Not done Probable USA300 + + 2 8IV Probable USA300 + 2 +8 IV MSSA Probable USA300 2 + 2 8 N/A Probable USA300 2 + + 8 N/A Non-USA300 MRSA + 22 8IV ++/2 +/2 8 Not SCCmec IV + 22Not ST8 Any ++2 Not ST8 Any + 2 + Not ST8 Any MSSA 2 +/2 +/2 Not ST8 N/A N/A, not applicable; SCCmec, staphylococcal cassette chromosome mec; ST, sequence type. 1120 IMMUNE RESPONSES AFTER S. AUREUS INFECTIONS

IL-6, IL-23, IL-10, IFN-g, IL-2, IL-17, IL-17F, and IL-22 in serum were presented to the ED following minor traumatic events (Fig. 1B). All measured by a multiplex beads assay (EMD Millipore, Billerica, MA). patients were treated with surgical incision and drainage unless they About 12 ml of blood containing sodium heparin anticoagulant was had nonpurulent cellulitis. Patients were randomized to one of two collected from all patients and uninfected controls. PBMCs from whole blood were isolated following Ficoll gradient centrifugation to eliminate antibiotics, clindamycin or trimethoprim/sulfamethoxazole, in pa- RBCs. These fresh PBMCs (106 cells/ml/well) were cultured in 48-well tients with multiple or large lesions, or to these antibiotics or a plates in a final volume of 1 ml, either unstimulated or stimulated with placebo when the abscess was small. Patients and controls had a anti-CD3 mAb (OKT3, 5 mg/ml), the TLR2 agonist Pam3CSK4 (1 mg/ml), similar distribution of gender, age, and self-reported race (Table II). or lysate from sonicated USA300 MRSA strain 923 (50 ml/well) for 24 h. PBMCs from frozen aliquots from a single healthy donor were used in Most individuals were African Americans, reflecting the population every plate as a positive control to verify the quality of the stimulation. The served by the ED at the University of Chicago, and ,40 y of age. supernatants were collected into Eppendorf tubes and frozen in aliquots Nasal, oropharyngeal, and perianal/inguinal cultures were obtained at 280˚C. The cytokine concentration of TNF, IL-1b, IL-6, IL-23, IL-10, to establish the colonization status of infected patients at D0 and IFN-g, IL-2, IL-17, and IL-22 was determined by ELISA (R&D System, D40 and uninfected controls at D0, and colonization was consid- Minneapolis, MN) according to the the manufacturer’s instructions, or, for IL-17A following USA300 MRSA sonicate restimulation by microfluidic ered positive when at least one of the sites tested positive. Colo- ELISA (Siloam Biosciences, Cincinnati, OH). nization was detected in similar proportions at all three body sites. Individuals were classified as uncolonized (with S. aureus)oras Statistical analysis colonized with USA300 versus non-USA300 S. aureus strain types. The numbers of infected patients and uninfected controls to enroll in the Isolates not genotyped were designated untyped (Fig. 1C). Fifty-six . study were determined by a sample size calculation to obtain 80% power percent of controls and 72% of patients on D0 were colonized with (with p , 0.05) in detecting a predetermined difference in the IL-17/IFN-g ratio between cases (median, 0.25; interquartile range, 0.12–0.050) and S. aureus, and within those, 19% of controls and 46% of SSTI Downloaded from controls (median, 0.43; interquartile range, 0.16–1.02) based on the dis- patients were colonized with USA300 (Fig. 1C). Analysis of col- tribution of cytokine production observed in preliminary results. The onization in patients on D40 revealed a decrease in colonization sample size was calculated forcing a ratio of cases to controls of 1:2 to prevalence that was not statistically significant (using the x2 test) increase power with a smaller number of cases. following antibiotic treatment, with 50% of patients colonized Statistical analyses on cytokines from PBMCs were performed using the nonparametric Mann–Whitney U test and GraphPadPrism 6.0 software. with S. aureus, among which 44% were USA300. Among

Analyses of serum cytokines were performed using a Wilcoxon rank-sum test, colonized individuals, 52.6, 51.9, and 39.4% were positive at http://www.jimmunol.org/ or using a Wilcoxon rank-sum test for matched pairs when appropriate. The more than one site in controls and infected patients at D0 and D40, 2 x test or the McNemar test for paired data was used to compare categorical respectively. Of note, some shifts in colonizing strains were ap- data. A p value ,0.05 was considered to be statistically significant. parent in infected patients between D0 and D40 (Table III) either because of the antibiotic treatment received by the patients or Results because shifts occur normally over time. Patient demographics, colonization, and infection status The infection isolate was identified in the clinical microbiology Overall, 215 adults were enrolled, including 72 patients with an laboratory and later sent to the MRSA Research Laboratory for SSTI and 143 controls (Fig. 1A, Table I). Most uninfected controls molecular typing and classification into USA300 or non-USA300 by guest on September 24, 2021

FIGURE 1. Enrollment, colonization, and infection status. (A) Schematic representation of enrollment among controls and SSTI patients. (B) Distri- bution of the chief complaint by uninfected controls presenting to the ED. (C) Enrolled individuals were swabbed at three body sites, and the colonization status and genotype of the S. aureus strain when at least one site was positive were determined at the ﬁrst visit to the ED for SSTI patients and uninfected controls (D0) as well as on D40 for SSTI patients. (D) Patients presenting with SSTI on D0 had their skin lesion incised and drained when possible and the obtained material was sent to the laboratory for genotyping. Patients whose samples grew S. aureus MRSA or MSSA in the clinical microbiology laboratory but that were not genotyped are labeled as untyped. Patients whose samples were not obtained or whose culture was negative are labeled as unknown. The Journal of Immunology 1121

Table II. Patient demographics sulfamethoxazole or those treated with clindamycin (Supplemental Fig. 1). These results support a systemic consequence of the lo- Controls Patients calized infection on D0, and they suggest that the SSTI results in Total numbers 143 72 either increased numbers of circulating cells that can respond to Gender (%) these stimuli, or in an increased capacity on a per cell basis to Female 56 54 produce these cytokines. Male 44 46 To investigate whether USA300 infection elicited distinct re- Age (%) 18–25 y 30 38 sponses compared with other S. aureus strain types, we compared 26–40 y 41 33 the production of innate cytokines by PBMCs from patients in- .41 29 29 fected with documented USA300 versus from patients infected Race (%) with documented non-USA300. As shown in Fig. 2B, PBMCs African American 92 97 from patients with documented USA300 infection produced more White 7 3 Asian ,10IL-1b and IL-6 upon restimulation with S. aureus sonicates, suggestive of a more robust systemic immune response. In contrast to the differences in innate cytokine production, no strain types. Eleven infection samples were identiﬁed as MRSA and differences were observed in the adaptive cytokines (IL-2, IFN-g, two as MSSA by the clinical microbiology laboratory but were not IL-17A, IL-22) detected following anti-CD3– or USA300 MRSA typed by the MRSA Research Laboratory and are labeled as MRSA- sonicate–mediated stimulation, either between SSTI patients and untyped or MSSA-untyped in Fig. 1D. Patients with nonpurulent uninfected controls (Supplemental Fig. 2A) or between SSTI pa- Downloaded from cellulitis that could not be drained (n = 10), negative cultures, and tients infected with USA300 versus non-USA300 (Supplemental samples that did not undergo typing (n = 16) were all combined into Fig. 2B), although signiﬁcantly higher IL-10 production was de- a category termed “unknown” for the infecting agent (Fig. 1D). All tected from USA300-infected versus non–USA300-infected pa- positive cultures revealed S. aureus as the infecting agent, and tients upon S. aureus restimulation (Supplemental Fig. 2B). 27.8% of SSTI patients were infected with documented USA300. Taken together, these results argue against the hypotheses that

Of interest, the infecting S. aureus strain was often different from infected patients express downmodulating polymorphisms that de- http://www.jimmunol.org/ the colonizing strain (Table IV), suggesting that distinct strains may crease their immune responsiveness to S. aureus strains, or that the colonize versus infect, or that by typing only one colony from each infection dampens immune responses. anatomic site, we missed detection of other colonizing strains that Serum cytokines were increased in infected patients may have included the infecting strain. The increased production of innate cytokines by PBMCs from Stimulated PBMCs from SSTI patients produced more innate infected patients compared with uninfected controls suggested that but not adaptive cytokines a systemic immune response was taking place in response to the Otherwise healthy individuals who develop S. aureus SSTIs may localized infection, at least in some patients. To further investigate be those with slightly lower global immune responses, which possible systemic consequences of the infection, the concentration by guest on September 24, 2021 might fail to contain colonizing bacteria or emerging infections. of serum cytokines was measured in uninfected controls and To test this hypothesis, we compared the innate cytokines produced SSTI patients. A higher proportion of infected patients on D0 had by PBMCs of SSTI patients during and after infection versus un- detectable serum cytokines compared with uninfected controls infected controls upon in vitro stimulation with the TLR2 agonist (Fig. 3A), and infected patients had signiﬁcantly higher serum levels Pam3CysK4, or with a USA300 MRSA sonicate, to mimic signals of TNF, IL-6, IL-10, IL-17F, and IL-22 (Fig. 3B). By D40, the elicited by S. aureus. Additionally, we measured the adaptive cy- serum IL-6 and IL-17F levels were signiﬁcantly decreased com- tokines produced following exposure to the pan–T cell activator pared with D0 (Fig. 3B), and similarly so in patients treated with anti-CD3 mAb, or to the USA300 MRSA sonicate. In contrast to trimethoprim/sulfamethoxazole or clindamycin (Supplemental Fig. our hypothesis, innate cytokine production by PBMCs obtained 1 for IL-6 and data not shown for IL-17F). The detectable level of from SSTI patients on D0 was actually increased (Il-1b, IL-6, and circulating cytokines was probably not due to systemic dissemina- TNF) when compared with that from control individuals, both tion of the infection, as a criterion for enrollment was a body following Pam3CysK4 and USA300 stimulation (Fig. 2A). In most temperature below 38.5˚C. In fact, all patients but four had a body cases, cytokine levels were lower when the infection was resolved temperature ,37˚C (Supplemental Fig. 3), and the four patients after treatment on D40, but not lower than from control PBMCs with a temperature between 37 and 37.9˚C did not have higher (Fig. 2A). Of note, similar cytokine level decreases between D0 and serum cytokine levels than did the afebrile patients (data not D40 were observed in infected patients treated with trimethoprim/ shown). Of note, SSTI patients infected with documented USA300

Table III. Colonizing S. aureus strains change over time

Patient Colonization on D40

Colonized

Patient Colonization on D0 Uncolonized USA300/MRSA Non-USA300/MRSA USA300/MSSA Non-USA300/MSSA N/A N/D Uncolonized (n = 20) 10 1 3 6 N/D (n =2) 1 1 USA300/MRSA (n = 18) 1 4 1 3 2 7 USA300/MSSA (n =5) 1 2 2 Non-USA300/MSSA (n = 27) 8 5 6 4 4 Total = 72 20 9 2 3 13 6 19 N/A, unknown; N/D, not done. 1122 IMMUNE RESPONSES AFTER S. AUREUS INFECTIONS

Table IV. Colonization status of SSTI patients with documented S. aureus infection

Colonization Status

D0 D40

Infecting Strain Uncolonized USA300 Non-USA300 Uncolonized USA300 Non-USA300 USA300 (n = 20) 7 8 5 8 5 2 Non-USA300 (n = 13) 2 6 5 3 1 3 Total 9 14 10 11 6 5

did not have greater serum cytokine levels than did those infected signiﬁcantly different in the colonized versus uncolonized control with documented non-USA300 (Supplemental Fig. 4), although the individuals (Fig. 4), suggesting that colonization does not desen- study was underpowered to analyze this parameter, as not all patients sitize the innate immune response to S. aureus in these individuals had measurable serum cytokines (Fig. 3A). and that a T cell repertoire capable of recognizing S. aureus Ags or superantigens is still present in the blood of colonized hosts. Impact of S. aureus colonization on immune responses Colonization also did not impact responses to Pam3CysK4 or anti- Our study design also allowed us to test the hypothesis that CD3 restimulation, or production of other cytokines tested (data Downloaded from asymptomatic colonization in uninfected individuals may tolerize not shown). immune cells, such that immune responses to S. aureus may be- Finally, we tested whether the type of S. aureus colonization come blunted, and colonized individuals may thus become more affected the immune responses in infected patients. Surprisingly, susceptible to infection either by the colonizing strain itself or by irrespective of the infecting strain, PBMCs from infected patients a subsequent strain to which they may become exposed. To test who were also colonized with USA300 on D0 produced more this hypothesis, we compared levels of cytokines produced upon in IL-17A, IL-22, and IL-10, but not IFN-g (or IL-2, not shown), vitro stimulation with USA300 MRSA sonicates by PBMCs from upon anti-CD3 or USA300 MRSA sonicate restimulation than did http://www.jimmunol.org/ noninfected controls that were either not colonized at the time of those colonized with non-USA300 strains, and those differences ED visit, or were colonized by USA300 versus non-USA300 dissipated after resolution of the infection on D40 (Fig. 5). strain types. Neither innate nor adaptive cytokine production was IL-17A, IL-22, and IL-10 are cytokines that have been associated by guest on September 24, 2021

FIGURE 2. Greater innate cytokines produced by stimulated PBMCs from SSTI patients than controls. (A) PBMCs from uninfected controls (Ctrl) and

SSTI-infected patients (Pt) were stimulated with Pam3CysK4 (Pam3, top panels) or with USA300 MRSA sonicates (bottom panels) for 24 h. Supernatants were assayed for IL-1b, IL-6, and TNF by ELISA. (B) Levels of cytokines induced by USA300 MRSA sonicates were compared between PBMCs from patients with documented non-USA300 infection and PBMCs from patients with documented USA300 infection. Plots represent mean 6 SEM. Statistical signiﬁcance was calculated using the Mann–Whitney U test. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 1123

FIGURE 3. Elevated serum cytokines in SSTI patients. Serum was isolated from SSTI patients (Pt) and uninfected controls (Ctrl) and cytokine levels were measured by multiplex bead assay. (A) Percentage of patients with detectable levels of given cytokines. Statistical signiﬁcance between uninfected and infected D0 was calculated using the x2 test and between D0 and D40 using the McNemar test for paired data. (B) Levels of selected cytokines are shown (mean 6 SEM). Sta- tistical signiﬁcance was calculated using the two-way ANOVA. *p , 0.05, **p , 0.01, ***p , 0.001. Downloaded from http://www.jimmunol.org/

with immune responses to S. aureus in vitro (35), suggesting that fected controls. In contrast to the hypothesis, we found that pa- we may be detecting a circulating memory T cell response in tients with SSTIs produced higher levels of innate cytokines than infected patients who are colonized with USA300. did uninfected controls, as determined in serum measurements as well as upon PBMC restimulation. Notably, skin infection with Discussion documented USA300 elicited greater innate cytokine production by guest on September 24, 2021 Skin infections constitute a public health concern. However, the by PBMCs than did skin infection with documented non-USA300. immune responses in patients with SSTIs are not well documented, Moreover, colonization with USA300, compared with non- and hypotheses to address why some individuals are susceptible to USA300 strain types, correlated with increased recall adaptive these infections have not been tested. In this study, we genotyped cytokine production by PBMCs of SSTI patients at the time of the colonizing and infecting S. aureus strains of patients with infection, but not after resolution of the infection or in uninfected SSTIs, analyzed their immune responses at D0 and D40 postin- controls. Our results argue against systemic tolerization by colo- fection, and tested whether the reason why otherwise healthy nizing strains, or inhibitory immune polymorphisms, as facilita- individuals develop SSTIs is because they have reduced global or tors of SSTIs in otherwise healthy individuals and, instead, S. aureus–speciﬁc immune responses when compared with unin- demonstrate a detectable systemic immune response in patients

FIGURE 4. Documented S. aureus colonization is not associated with reduced PBMC responses in uninfected subjects. PBMCs from uninfected controls were stimulated with USA300 MRSA sonicates for 24 h, and cytokine levels in supernatants were measured by ELISA. The plots compare the cytokines produced by uninfected controls classified as uncolonized versus those classified as colonized with USA300 and those classified as colonized with non-USA300 genotypes (mean 6 SEM). Differences were not significant as determined by the Mann–Whitney U test. 1124 IMMUNE RESPONSES AFTER S. AUREUS INFECTIONS

FIGURE 5. PBMCs from SSTI patients colonized with USA300 produce more IL-17, IL-22, and IL-10 cytokines than do those from SSTI patients colonized with non-USA300. PBMCs from SSTI patients were stimulated for 24 h with USA300 MRSA sonicates (top panels) or anti-CD3 (bottom panels) and cytokine levels in the supernatants were determined by ELISA. Results from patients with documented colonization with USA300 were compared with those from patients with documented colonization with non-USA300 S. aureus strain types. Plots show mean 6 SEM. *p , 0.05, **p , 0.01, ***p , Downloaded from 0.001 as determined by the Mann–Whitney U test. with localized, uncomplicated SSTIs, with greater systemic im- previously found that 25% of individuals of the combined adult and mune responses when USA300 was detected either as the patho- pediatric population enrolled in the parental study were polyclonally

gen in an SSTI or as a commensal in the cultures obtained to test colonized (27). Consistent with the concept of polycolonization, we http://www.jimmunol.org/ for colonization. also found that the infecting S. aureus isolates often differed geno- The prevalence of S. aureus colonization observed in the adult typically from isolates that colonized the same individual, indicating population enrolled in this study, namely 56% of controls and 72% that many patients are infected with one strain and simultaneously of patients, matched that obtained in the parent study, which in- colonized with another. Whether the infecting strain is also present cluded both adult and pediatric subjects (27), but it was higher as a colonizing commensal at another site of the body in these pa- than that usually cited in the general population when only the tients or has been present as a colonizing commensal before the nares are sampled (36). Because we considered patients to be infection remains to be determined. colonized when S. aureus was isolated from any of three body S. aureus was the only infectious agent identified in positive sites cultured, and because analysis of only the nares swabs cultures from SSTI lesions in our patients. Although we cannot by guest on September 24, 2021 yielded similar percentages to previous reports (27), our data exclude that the nonpurulent cellulitis that could not be drained, suggest that S. aureus colonization is more frequent than com- or lesions with negative cultures, were caused by non–S. aureus monly recognized and more widespread throughout the body microbial organisms, it is reasonable to speculate that the vast (27). People become colonized with limited microbiota at birth, majority of SSTI lesions are caused by S. aureus. Thus, when and staphylococci have been shown to be among the first colo- analyzing the data, we first grouped all infected patients into a nizing taxa (37). The demonstration that isotype-switched anti– single category regardless of the results of cultures from their S. aureus Abs can be rapidly and increasingly detected during SSTI site. childhood, in the absence of obvious infection (38), implies rapid Our data reveal increased innate cytokines in infected patients general exposure and generation of adaptive immunity to coloniz- compared with controls. Although the differences in mean cytokine ing S. aureus strains or cross-reacting strains. This is also consistent production between the groups were small, the statistically sig- with a recent report identifying a high level of T cell memory to nificant increase in innate cytokine levels in infected patients on D0 S. aureus in healthy individuals, with the S. aureus–specific T cell when compared with uninfected controls correlated with the on- frequency estimated between 0.2 and 5.7% of the T cell repertoire going infection, and the significant reduction on D40 compared (4). Taken together, these studies suggest that healthy individuals with D0 (and the lack of statistical difference on D40 with unin- may in fact not be immunologically naive to S. aureus Ags. fected controls) correlated with resolution of the infection, sug- The epidemiologic data from our study demonstrates that gesting that, even if subtle, these differences were associated with a USA300 colonization was more frequent in SSTI patients than in functional response. Although it is unclear whether the higher controls, and that antibiotic treatment failed to clear S. aureus cytokine levels in patients infected with USA300 compared with carriage in many patients, irrespective of its genotype and despite non-USA300 are biologically meaningful, this stronger immune resolution of the skin infection in most cases. Moreover, the ge- response may be necessary to clear the infection by such a virulent notype of colonizing strains changed from D0 to D40 in many strain, or may result in more severe inflammatory symptoms that patients. This may be due to pressure imparted by the antibiotics then bring more patients to the ED, which, in turn, may explain the administered to treat the infection and/or to shifts that may occur detection of this strain type among SSTI cultures in epidemic over time even in healthy controls not deliberately exposed to proportions. antimicrobial therapy. Alternatively, people may often be colo- Our results also reveal increased recall responses in infected nized by multiple genotypes simultaneously, such that sampling patients colonized with USA300. Recall memory T cell responses single colonies at two different time points may simply reveal two to S. aureus have been shown to reside in circulating CCR6+ distinct strain types that were both present throughout the time CCR4+ T cells that comprise Th17 as well as some Th1 cells, course. Indeed, even with the limited approach of genotyping only rather than in the CXCR3+ or CCR4+ subsets, which are enriched one bacterial colony from each of the three cultured sites, we in Th1 and Th2 cells, respectively (35). Differentiation of T cells The Journal of Immunology 1125 from healthy controls for 12 d in vitro with heat-inactivated 5. Brown, A. F., J. M. Leech, T. R. Rogers, and R. M. McLoughlin. 2014. Staphy- S. aureus lococcus aureus colonization: modulation of host immune response and impact on –pulsed monocytes revealed production of IL-17, IL-22, human vaccine design. Front. Immunol. 4: 507. doi:10.3389/fimmu.2013.00507. and IL-10 predominantly (35). Strikingly, IL-17, IL-22, and IL-10 6. Maro´di, L., S. Cypowyj, B. Toth, L. Chernyshova, A. Puel, and J. L. Casanova. were also the cytokines whose 24 h recall production we found to be 2012. Molecular mechanisms of mucocutaneous immunity against Candida and Staphylococcus species. J. Allergy Clin. Immunol. 130: 1019–1027. doi:10.1016/ significantly increased in PBMCs from SSTI patients colonized with j.jaci.2012.09.011. USA300, irrespective of the infecting strain’s genotype. These cy- 7. Stappers, M. H., Y. Thys, M. Oosting, T. S. Plantinga, M. Ioana, P. Reimnitz, tokines can induce tissue-protective and immunosuppressive effects, J. W. Mouton, M. G. Netea, L. A. Joosten, and I. C. Gyssens. 2014. Polymor- phisms in cytokine genes IL6, TNF, IL10, IL17A and IFNG influence suscepti- and they have been implicated at cutaneous sites (35). The increased bility to complicated skin and skin structure infections. Eur. J. Clin. Microbiol. capacity of circulating PBMCs to produce these cytokines upon Infect. Dis. 33: 2267–2274. doi:10.1007/s10096-10014-12201-10090. in vitro recall may reflect an additive Th17-type effector/memory 8. Stappers, M. H., M. Oosting, M. Ioana, P. Reimnitz, J. W. Mouton, M. G. Netea, I. C. Gyssens, and L. A. Joosten. 2015. Genetic variation in TLR10, an inhibitory response to the ongoing infection and USA300 colonization. This is Toll-like receptor, influences susceptibility to complicated skin and skin struc- supported by the fact that colonization with USA300 did not result ture infections. J. Infect. Dis. 212: 1491–1499. doi:10.1093/infdis/jiv229. in increased T cell recall responses in uninfected controls. 9. Fleischer, B. 1994. Superantigens produced by infectious pathogens: molecular mechanism of action and biological significance. Int. J. Clin. Lab. Res. 24: 193–197. We also tested whether exposure of a person’s immune system to 10. Hedl, M., J. Li, J. H. Cho, and C. Abraham. 2007. Chronic stimulation of Nod2 asymptomatically colonizing S. aureus could dampen immune re- mediates tolerance to bacterial products. Proc. Natl. Acad. Sci. USA 104: 19440– 19445. doi:10.1073/pnas.0706097104. sponses to S. aureus Ags and microbial signals, thereby enhancing 11. Hruz, P., A. S. Zinkernagel, G. Jenikova, G. J. Botwin, J. P. Hugot, M. Karin, susceptibility of the person to infection. However, our analysis of the V. Nizet, and L. Eckmann. 2009. NOD2 contributes to cutaneous defense against uninfected control subjects with detectable but asymptomatic Staphylococcus aureus through alpha-toxin-dependent innate immune activation. Proc. Natl. Acad. Sci. USA 106: 12873–12878. doi:10.1073/pnas.0904958106. S. aureus colonization did not reveal any decrease in the ability of 12. Harrison, C. J. 2009. Innate immunity as a key element in host defense against Downloaded from their PBMCs to produce innate or adaptive cytokines upon S. aureus methicillin resistant Staphylococcus aureus. Minerva Pediatr. 61: 503–514. restimulation when compared with uncolonized controls. This result 13. Puel, A., C. Picard, M. Lorrot, C. Pons, M. Chrabieh, L. Lorenzo, M. Mamani- Matsuda, E. Jouanguy, D. Gendrel, and J. L. Casanova. 2008. Recurrent staph- should be interpreted with caution because it is possible, and perhaps ylococcal cellulitis and subcutaneous abscesses in a child with autoantibodies likely, that all individuals are colonized with S. aureus either tran- against IL-6. J. Immunol. 180: 647–654. 14. Spellberg, B., A. S. Ibrahim, M. R. Yeaman, L. Lin, Y. Fu, V. Avanesian, siently or persistently, such that the uninfected subjects who we A. S. Bayer, S. G. Filler, P. Lipke, H. Otoo, and J. E. Edwards, Jr. 2008. The characterized as uncolonized may have in fact been colonized and antifungal vaccine derived from the recombinant N terminus of Als3p protects http://www.jimmunol.org/ already downregulated their immune responses to S. aureus. mice against the bacterium Staphylococcus aureus . Infect. Immun. 76: 4574–4580. doi:10.1128/IAI.00700-08. In conclusion, our results do not support the hypothesis that SSTIs 15. Lin, L., A. S. Ibrahim, B. Baquir, V. Avanesian, Y. Fu, and B. Spellberg. 2009. occur in individuals with more blunted systemic immune responses Immunological surrogate marker of rAls3p-N vaccine-induced protection against when compared with people who do not develop infections, but rather Staphylococcus aureus. FEMS Immunol. Med. Microbiol. 55: 293–295. 16. Ishigame, H., S. Kakuta, T. Nagai, M. Kadoki, A. Nambu, Y. Komiyama, that ongoing USA300 skin infections drive a stronger innate in- N. Fujikado, Y. Tanahashi, A. Akitsu, H. Kotaki, et al. 2009. Differential roles of flammatory response than do non-USA300 skin infections, whereas interleukin-17A and -17F in host defense against mucoepithelial bacterial in- USA300 colonization may induce stronger Th17 memory. A limi- fection and allergic responses. Immunity 30: 108–119. 17. Chan, L. C., S. Chaili, S. G. Filler, K. Barr, H. Wang, D. Kupferwasser, tation of our study is that only systemic immune responses were J. E. Edwards, Jr., Y. Q. Xiong, A. S. Ibrahim, L. S. Miller, et al. 2015. Nonre- analyzed. Although our data show that there are no defects in global dundant roles of interleukin-17A (IL-17A) and IL-22 in murine host defense by guest on September 24, 2021 or S. aureus–specific T cell immunity, it remains possible that against cutaneous and hematogenous infection due to methicillin-resistant Staphylococcus aureus. Infect. Immun. 83: 4427–4437. doi:10.1128/IAI.01061-15. S. aureus–reactive T cells may be impaired in their migration to the 18. Cho, J. S., E. M. Pietras, N. C. Garcia, R. I. Ramos, D. M. Farzam, skin, or that the infection milieu may directly suppress the function H. R. Monroe, J. E. Magorien, A. Blauvelt, J. K. Kolls, A. L. Cheung, et al. 2010. IL-17 is essential for host defense against cutaneous Staphylococcus aureus of these T cells following cutaneous infiltration. Local immune re- infection in mice. J. Clin. Invest. 120: 1762–1773. doi:10.1172/JCI40891. sponses to skin commensals and pathogens are only starting to be 19. Wolk, K., K. Warszawska, C. Hoeflich, E. Witte, S. Schneider-Burrus, K. Witte, investigated (39) and may reveal additional insights into suscepti- S. Kunz, A. Buss, H. J. Roewert, M. Krause, et al. 2011. Deficiency of IL-22 contributes to a chronic inflammatory disease: pathogenetic mechanisms in acne bility to SSTIs. Our study also raises additional questions for sub- inversa. J. Immunol. 186: 1228–1239. doi:10.4049/jimmunol.0903907. sequent studies, including why pre-existing T cell responses do not 20. Montgomery, C. P., M. Daniels, F. Zhao, M. L. Alegre, A. S. Chong, and protect from infection, what role these immune responses play in the R. S. Daum. 2014. Protective immunity against recurrent Staphylococcus aureus skin infection requires antibody and interleukin-17A. Infect Immun. 82: 2125– clearing of the infection and in tissue pathology, and what genetic 2134. doi:10.1128/IAI.01491-14. traits present in USA300, but not other S. aureus strain types, drive 21. Holland, S. M., F. R. DeLeo, H. Z. Elloumi, A. P. Hsu, G. Uzel, N. Brodsky, stronger immune responses. A. F. Freeman, A. Demidowich, J. Davis, M. L. Turner, et al. 2007. STAT3 mutations in the hyper-IgE syndrome. N. Engl. J. Med. 357: 1608–1619. 22. Ma, C. S., G. Y. Chew, N. Simpson, A. Priyadarshi, M. Wong, B. Grimbacher, Acknowledgments D. A. Fulcher, S. G. Tangye, and M. C. Cook. 2008. Deficiency of Th17 cells in hyper IgE syndrome due to mutations in STAT3. J. Exp. Med. 205: 1551–1557. 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pg/ m -2 (

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Supplemental Figure 1. The reduction in cytokine levels in serum and supernatants of stimulated PBMCs between D0 and D40 was similar in patients treated with trimetoprim/sulfamethoxazole and clindamycin. The difference in cytokine levels between D0 and D40 was measured for serum IL-6 (top left panel) and supernatants of USA300 sonicate-restimulated PBMCs for IL-6, IL-1β and TNF (other 3 panels). The plots represent the mean - SEM from patients treated with trimetoprim/sulfamethoxazole versus clindamycin (n=28/group). No statistical differences were found between the 2 groups. A Stimulation of PBMCs from uninfected controls and SSTI patients:

IL-2 IFN-γ IL-17A IL22 IL10 ) ) ) ) l l l l

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l 800 3000 2500 3000 m m m m

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Anti-CD3 1500 ( ] 400

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Sonicate 2 ] - γ ] (pg/ml) 22 ] 10 ] - 1000 1000 17 ] - 1000 - N 200 - L I L 20 L I I F L [ 500 I I [ [ [ 0 0 [ 0 0 Infecting strain: non-USA300 USA300 non-USA300 USA300 non-USA300 USA300 0 non-USA300 USA300 non-USA300 USA300

Supplemental Figure 2. Adaptive cytokines are similar is SSTI patients and controls and in SSTI patients infected with non-USA300 and USA300 S. aureus strain types. A. PBMCs from uninfected controls and infected patients were stimulated with anti-CD3 or USA300 MRSA sonicates and production of adaptive cytokines was measured in the supernatant at 24h by ELISA. B. Adaptive cytokines produced by PBMCs from SSTI patients with documented USA300 and non-USA300 infection were compared. Plots show Mean + SEM. *p<0.05, Mann- Whitney. Body Temperature 40 ) C

o 39 (

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Supplemental Figure 3. Body temperature of SSTI patients. Body temperature was measured in infected patients on D0. TNF IL-6 IL-10 10 60 NS NS 1.5 NS )

) 8 l ) l l m m m 40 6 1.0 pg / pg / pg / ( ( (

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Supplemental Figure 4. Serum cytokines are similar is SSTI patients infected with non-USA300 and USA300 S. aureus strain types. Serum from SSTI patients on D0 was analyzed for cytokine content by multiplex and cytokine levels were compared between patients infected with documented non-USA300 (n=12) versus USA300 (n=20) S. aureus strain types. NS, not significant.