Staphlyococcus aureus Phenol-Soluble Modulins Stimulate the Releaseof Proinflammatory Cytokines from Keratinocytes and Are Requiredfor Induction of Skin Inflammation

Adnan K. Syed,a,b Tamra J. Reed,c Kaitlyn L. Clark,c Blaise R. Boles,b J. Michelle Kahlenbergc

Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USAa; Department of Microbiology, Roy J. and Lucille A.Carver College of Medicine, University of Iowa, Iowa City, Iowa, USAb; Department of Internal Medicine, Division of Rheumatology, University of Michigan, Ann Arbor,Michigan, USAc

Staphylococcus aureus is a human commensal that colonizes the skin. While it is normally innocuous, it has strong associationswith atopic dermatitis pathogenesis and has become the leading cause of skin and soft tissue infections in the United States. Thefactors that dictate the role of S. aureus in disease are still being determined. In this work, we utilized primary keratinocyte cul-ture and an epidermal murine colonization model to investigate the role of S. aureus phenol-soluble modulins (PSMs) in proin-flammatory cytokine release and inflammation induction. We demonstrated that many species of Staphylococcus are capable ofcausing release of interleukin 18 (IL-18) from keratinocytes and that S. aureus PSMs are necessary and sufficient to stimulateIL-18 release from keratinocytes independently of caspase 1. Further, after 7 days of epicutaneous exposure to wild-type S. au-reus, but not S. aureus �psm, we saw dramatic changes in gross pathology, as well as systemic release of proinflammatory cyto-kines. This work demonstrates the importance of PSM peptides in S. aureus-mediated inflammatory cytokine release from kera-tinocytes in vitro and in vivo and further implicates PSMs as important contributors to pathogenesis.

Staphylococcus aureus is a human commensal that lives in thenose, skin, and throat in approximately 30% of the human

population (1, 2). While S. aureus is usually harmless, it is anopportunistic pathogen that has become a leading cause of noso-comial infections in the United States (3) and can manifest as skinand soft tissue infections, infective endocarditis, osteomyelitis,and sepsis (1). Further, it has a role in exacerbation of atopicdermatitis (AD), a chronic inflammatory disease of the skin thataffects up to 20% of children and up to 3% of adults (4, 5).Chronic cutaneous inflammation during AD results in increasedproduction of extracellular matrix components that permit at-tachment of S. aureus (6). Subsequently, S. aureus promotes ADby stimulating a strong proinflammatory response (7). S. aureusproteins, such as hemolysin � (HLA), staphylococcal protein A(SPA), and lipoteichoic acids (LTA), promote proinflammatorycytokine production from keratinocytes (7–11). However, thisstimulation requires the concurrent presence of a surfactant, suchas SDS (11).

Keratinocytes serve as the first line of defense against cutane-ous pathogens and are able to stimulate an immune response byreleasing cytokines, defensins, and antimicrobial cationic pep-tides, such as LL-37, to fight off pathogens (12). One cytokine thatis produced by keratinocytes and has a role in promoting AD isinterleukin 18 (IL-18) (13, 14). IL-18 is a proinflammatory cyto-kine that is cleaved and activated primarily by caspase 1 (15).Caspase 1 activation occurs following exposure to danger-associ-ated molecular patterns (DAMPs) or pathogen-associated molec-ular patterns (PAMPs), which stimulate the activation of the in-flammasome (16). Caspase 1 activation can also result in theactivation of the proinflammatory cytokine IL-1� (17). These ma-ture cytokines are then released from the cell and have variedeffects, such as leukocyte recruitment, promotion of T and B cellactivation, and upregulation of other inflammatory cytokines(18).

The phenol-soluble modulins (PSMs) of S. aureus have beenshown to have many characteristics, including the ability to re-cruit leukocytes, lyse erythrocytes and neutrophils, act as antimi-crobial peptides, modulate biofilm development, and exhibit sur-factant-like properties (19–28). PSMs are small peptides rangingfrom 22 to 44 amino acids in length that are predicted to formamphipathic helices and in some cases can form amyloid fibers toabrogate their toxic properties and stabilize biofilms (19, 23). Sev-eral staphylococcal species have been reported to produce PSMproteins, including S. aureus, Staphylococcus epidermidis, andStaphylococcus lugdunensis (19, 29, 30).

Here, we set out to investigate staphylococcal factors that con-tribute to the release of proinflammatory cytokines from kerati-nocytes. We found that some, but not all, species of staphylococciexamined produce compounds that are able to cause the lytic re-lease of active IL-18 from human keratinocytes independently ofcaspase 1 activation. We also showed that PSMs are necessary andsufficient for this release. Further, we utilized a mouse model ofcutaneous colonization (31) to demonstrate that neutrophil re-

Received 25 March 2015 Returned for modification 16 April 2015Accepted 10 June 2015

Accepted manuscript posted online 15 June 2015

Citation Syed AK, Reed TJ, Clark KL, Boles BR, Kahlenberg JM. 2015. Staphlyococcusaureus phenol-soluble modulins stimulate the release of proinflammatorycytokines from keratinocytes and are required for induction of skin inflammation.Infect Immun 83:3428 –3437. doi:10.1128/IAI.00401-15.

Editor: A. Camilli

Address correspondence to J. Michelle Kahlenberg, mkahlenb@med.umich.edu.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00401-15

3428 iai.asm.org September 2015 Volume 83 Number 9Infection and Immunity

on March 2, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

on March 2, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

on March 2, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

cruitment and systemic inflammatory response are dependent onS. aureus PSMs.

MATERIALS AND METHODSBacterial strains and growth conditions. The bacteria used in this studyare summarized in Table 1. S. aureus strain LAC (32) was used as thewild-type (WT) strain for the study. To create a �psm deletion strain, thePSM� and PSM� operons were deleted using allelic replacement, as pre-viously described (23), and the start codon of �-toxin was mutated toprevent translation without affecting the function of RNAIII, as was donepreviously (19). Genetic complementation of the PSM� and PSM� oper-ons in the �psm strain was done as previously described using pALC2073-PSM� and pRS-PSM� (23). Bacterial supernatants were collected after 24h of growth in tryptic soy broth (TSB) (MP Biosciences, Santa Ana, CA) at37°C with orbital shaking at 200 rpm. The cells were centrifuged at7,000 � g for 10 min, followed by filter sterilization of the supernatantsthrough 0.22-�m filters. The supernatants were stored at �20°C until use.Bacteria used for colonization were grown overnight in TSB at 37°C withorbital shaking at 200 rpm. Before use, they were diluted 1:500 in freshTSB and grown for 4 h to obtain cells in the exponential growth phase. Thecells were resuspended in phosphate-buffered saline (PBS) (Gibco, GrandIsland, NY) prior to use.

Peptides. PSM peptides were synthesized by LifeTein (South Plain-field, NJ) and assayed to be 90% pure by high-performance liquid chro-matography (HPLC). Synthetic peptides were prepared as previouslydescribed (33). Briefly, the peptides were resuspended in hexafluoroiso-propanol (HFIP), and 0.5 mg was aliquoted into microcentrifuge tubes.The HFIP was then removed using a SpeedVac system at room tempera-ture. The dried peptides were stored at �20°C. Immediately before use,the peptides were suspended in PBS to a concentration of 10 mg/ml andthen diluted further in PBS to the desired concentration. LL-37 was pur-chased from AnaSpec Inc. (Fremont, CA).

Cell culture. Primary human keratinocyte cultures were obtainedfrom Johann Gudjonsson, University of Michigan. The cells were culturedin EpiLife medium (Gibco) supplemented with human keratinocytegrowth supplement (Gibco), 1% Pen-Strep (100 U/ml penicillin and 100�g/ml streptomycin; Gibco), and 0.25 �g/ml amphotericin B (Fungizone;Gibco). For all experiments, cells were used at passages 2 to 6, and 2 � 104

cells/well were plated in 24-well plates.Cells were exposed to bacterial supernatants or peptides at confluence.

The cells were pretreated for 30 min with 10 �M caspase inhibitors (Enzo)where indicated prior to addition of the indicated concentrations of bac-terial supernatants or peptides. The cells were treated for 1 h prior to

harvesting. The extracellular medium was removed after 1 h, and theremaining cells were lysed using RIPA buffer (300 mM NaCl, 50 mM Tris,6.4 mM EDTA, 0.5% Triton X-100) containing protease inhibitor cocktail(Complete Mini, EDTA free; Roche, South San Francisco, CA) by incu-bation for 10 min on ice. The cell debris was removed by centrifugation at13,000 � g for 5 min, and the lysates were frozen at �20°C until use.

For caspase 1 activation studies, keratinocytes were plated at 1 � 104

cells/well of a 96-well plate. The following day, the cells were exposed to S.aureus supernatants for 1 h, followed by addition of fluorescein isothio-cyanate (FITC)-conjugated YVAD (ImmunoChemistry Technologies)for 1 h, and processed according to the manufacturer’s recommendations.The cells were counterstained with DAPI (4=,6-diamidino-2-phenylin-dole) before fixation. Images of active caspase 1 were acquired on anOlympus IX70 inverted microscope (Olympus, Center Valley, PA) using a40� objective at the Center for Live Cell Imaging (CLCI) at the Universityof Michigan Medical School.

To control for caspase inhibitor effectiveness, human peripheral bloodmononuclear cells (PBMCs) were isolated via Ficoll gradient. The cellswere plated at 1 � 106 per well of a 12-well plate; 10 �M YVAD or ZVADwas added 30 min prior to stimulation with 1 �g/ml lipopolysaccharide(LPS) for 24 h. The medium was collected, and IL-1� release was mea-sured via enzyme-linked immunosorbent assay (ELISA).

Mice. All animal studies were performed according to protocols ap-proved by the University of Michigan Committee on the Use and Care ofAnimals, protocol 01823. The animals were obtained from Jackson Lab-oratory and housed at the University of Michigan in specific-pathogen-free housing, followed by biohazard containment after treatment withbacterial strains. To test the effects of exposure to WT S. aureus versus S.aureus �psm, the dorsal skin of 8- to 10-week-old C57BL/6 female micewas shaved and depilated with Veet (Reckitt Benckiser Group plc). Thenext day, the stratum corneum barrier was disrupted via mild strippingusing three applications of Tegaderm (3M, St. Paul, MN). This techniqueexposes the keratinocyte layers without creating a wound (31). S. aureus(1 � 107 CFU) or PBS was placed with sterile gauze on the stripped skinand occluded with Tegaderm (3M). One week after S. aureus exposure,the animals were euthanized. Serum was collected via cardiac puncture,and skin was removed for cell count analysis via flow cytometry (see be-low) and RNA analysis.

Cytokine production. In vitro quantification of IL-18 and IL-1� wasdone by ELISA (eBioscience, Vienna, Austria) according to the manufac-turer’s instructions. Active IL-18 release was assessed via Western blotting(primary antibody, rabbit anti-human IL-18 [Santa Cruz]; secondary an-tibody, goat anti-rabbit-horseradish peroxidase [HRP] [Abcam]). To in-vestigate changes in the serum cytokine profiles of mice exposed to S.aureus, a Milliplex (EMD Millipore, Darmstadt, Germany) assay was per-formed.

Cytotoxicity. To test for cell lysis following peptide treatments of ke-ratinocytes, release of lactate dehydrogenase (LDH) was measured using aCytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison,WI) according to the manufacturer’s instructions.

Flow cytometry. Lymphocytes were isolated from skin biopsy speci-mens as follows. Immediately after euthanasia, skin samples were re-moved, diced into 1- to 2-mm pieces using sterile razor blades, and thenplaced into a GentleMacs C Tube with 10 ml RPMI-10 (RPMI plus 10%fetal bovine serum) with 2 ml enzyme solution (100 mg/ml DNase type I[Sigma; DN-25], 100 mg/ml hyaluronidase type V [Sigma; H6254], 500mg/ml collagenase [Sigma; C5138] in Hanks balanced salt solution[HBSS]). GentleMacs tissue dissociation was performed for 1 min, fol-lowed by 2 h of incubation at 37°C. After incubation, dissociation wasrepeated for 1 min. The cells were strained and pelleted by centrifugationat 800 � g for 7 min at 4°C. The supernatant was discarded, and the cellswere washed once with 15 ml RPMI-10. After washing, the cells weredivided into tubes for fluorescence-activated cell sorter (FACS) analysis.To quantify the inflammatory cellular infiltrate following S. aureus expo-sure, purified cells were stained for 1 h on ice with the following antibodies

TABLE 1 Bacterial strains used in this study

Strain Description or speciesSource orreference

S. aureusAH1181 LAC hla::erm This studyAH1263 LAC (WT) 32AH1292 LAC agr::tet 47BB3047 LAC �psm; pALC2073 and pAH8 This study, 23BB3048 LAC �psm; pALC2073-PSM�

and pRS-PSM�This study, 23

Other staphylococciBB2153 S. saprophyticus This studyBB2164 S. epidermidis This studyBB2191 S. lugdunensis This studyBB2201 S. warneri This studyBB2203 S. hominis This studyBB2205 S. haemolyticus This studyBB2207 S. capitis This study

S. aureus PSMs Promote Skin Inflammation

September 2015 Volume 83 Number 9 iai.asm.org 3429Infection and Immunity

on March 2, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

(all from BioLegend, San Diego, CA): CD19-phycoerythrin (PE)-Cy7,CD3-allophycocyanin (APC), CD4-FITC, CD8-PE, Ly6G-FITC, F4/80Pacific Blue, CD11b-APC, CD11c-PE-Cy7, CD11c-PE, and Ly6C-PE-Cy7. Flow data were collected on a BD LSR II and analyzed with FlowJoV10 (Tree Star).

RT-PCR. Skin biopsy specimens were homogenized in TriPure(Roche), and RNA was isolated with Direct-zol mini RNA prep (Zymo).RNA (100 ng) was transcribed into cDNA, and real-time (RT) PCR anal-ysis was completed on an ABI Prism 7900HT (Applied Biosystems) usingSybr green (Life Technologies) (Table 2 lists the primers). Cycle timeswere normalized to �-actin, and the fold change (2���CT) was calculatedversus PBS-exposed mice.

Statistical analysis. Statistical analysis was performed using Graph-Pad Prism 6 software. Comparisons between data were made via 2-sidedStudent unpaired t tests or Mann-Whitney U tests for nonnormalizeddata. A P value of 0.05 was considered significant.

RESULTSSome staphylococci produce cytotoxic factors that affect humankeratinocytes. To investigate the effects that secreted factors fromdifferent staphylococci have on human keratinocytes, we exposedhuman keratinocytes to bacterial supernatants. The keratinocyteswere monitored for the release of IL-18 because of its constitutiveproduction in keratinocytes and its proinflammatory activity inthe skin (11). We found that supernatants from several staphylo-coccal species, including S. aureus, S. lugdunensis, S. epidermidis,Staphylococcus capitis, Staphylococcus warneri, and, to a lesser ex-tent but still significantly, Staphylococcus hominis, were able totrigger the release of IL-18 from keratinocytes (Fig. 1A). Superna-tants from Staphylococcus saprophyticus and Staphylococcus hae-molyticus did not induce release of IL-18 (Fig. 1A). Importantly,release of LDH, a marker of cell lysis, aligned with IL-18 releasefrom the cells, with the exception of S. hominis, which showed nosignificant increase in LDH release (Fig. 1B).

S. aureus phenol-soluble modulins induce IL-18, IL-1�, andLDH release from human keratinocytes. Because of its promi-nent role in skin infections and AD, we chose to focus the rest ofour study on S. aureus. Using a candidate approach, we found thatthe quorum-sensing system in S. aureus known as the accessorygene regulator (agr) was necessary for cytotoxicity and IL-18 and

IL-1� release (Fig. 1C, D, and E). Because the agr system is knownto regulate the expression of many virulence factors produced byS. aureus, we investigated mutants for two well-studied cytotoxicfactors: HLA and the PSM family. S. aureus supernatants lackingPSMs (PSM� [PSM�1 to -4], PSM� [PSM�1 and -2], and�-toxin) (�PSM) showed a significant decrease in the ability tostimulate the release of IL-18, IL-1�, and LDH from keratinocytes,and the release was restored when the strains were complementedwith PSM� and PSM� operons (Fig. 1C, D, and E). Notably, lackof HLA, which has been reported to lyse human cells and activatethe inflammasome (34), did not impact the release of IL-18, LDH,or IL-1� compared to our WT S. aureus supernatant (Fig. 1C, D,and E). These data suggest that PSMs are the primary S. aureusfactors that induce lysis of keratinocytes.

To further investigate which PSMs contribute to IL-18 releaseand keratinocyte lysis, synthetic PSMs were used. PSM peptideswere sufficient to stimulate the release of IL-18 from keratinocytes(Fig. 2A). Interestingly, all of the PSM� peptides, as well as�-toxin, but neither of the PSM� peptides, induced release ofLDH from the keratinocytes (Fig. 2B). As PSMs are thought tohave a helical structure similar to that of the known lytic antimi-crobial peptide LL-37 (reviewed in reference 35), we also com-pared the dose response of the ability of PSM�1 to induce IL-18release to that of LL-37. As shown in Fig. 2C, both peptides in-duced IL-18 release at 5 �g/ml, but LL-37 appears to be morepotent. Taken together, these data demonstrate that the PSM pep-tides of S. aureus are necessary and sufficient for the release ofIL-18, likely through lytic release from human keratinocytes.

The release of mature IL-18 is independent of caspase activ-ity. Several S. aureus toxins have been reported to activate theinflammasome, so we next investigated whether inflammasomeactivation participated in the release of IL-18 from keratinocytesfollowing S. aureus exposure (36). As shown in Fig. 3A, we con-firmed that stimulation of keratinocytes with WT S. aureus super-natant resulted in detectable active caspase 1 within the keratino-cytes. Western blotting confirmed that the IL-18 released was in itsactive form (Fig. 3B). To see if caspase 1 activation was requiredfor the release of IL-18 or IL-1�, we pretreated primary keratino-

TABLE 2 Primers used for real-time PCR

Gene

Primer (5=¡3=)

Forward Reverse

IFN-� ATGGCTAGRCTCTGTGCTTTCCT AGGGCTCTCCAGAYTTCTGCTCTGIFN-� AGCTCCAAGAAAGGACGAACAT ATTCTTGCTTCGGCAGTTACIFN-� AGCGGCTGACTGAACTCAGATTGTA GTCACAGTTTTCAGCTGTATAGGGIL-18 ACTGTACAACCGCAGTAATACGC AGTGAACATTACAGATTTATCCCIL-1� CCCTGCAGCTGGAGAGTGTGGA CTGAGCGACCTGTCTTGGCCGIL-6 CTGCAAGAGACTTCCATCCAG AGTGGTATAGACAGGTCTGTTGGIL-1� CGGGTGACAGTATCAGCAAC GACAAACTTCTGCCTGACGATNF-� CCCACTCTGACCCCTTTACT TTTGAGTCCTTGATGGTGGTCAMP TCAACCAGCAGTCCCTAGAC AAGGCACATTGCTCAGGTAGNLRP3 ATGCTGCTTCGACATCTCCT AACCAATGCGAGATCCTGACChemokine (C-C motif) receptor 1 (CCR1) TGGGCAATGTCCTAGTGATT GCATCACCAAAAATCCAGTCCCL4 AGCAACACCATGAAGCTCTG CTGTCTGCCTCTTTTGGTCACCL5 CAATCTTGCAGTCGTGTTTG GGAGTGGGAGTAGGGGATTAC-X-C motif chemokine 10 (CXCL10) CAAAAGTAACTGCCGAAGCA CTGAGCTAGGGAGGACAAGGCXCL13 AGAGGTTTGCGAGATGGACT GAGCCTGGACCTTTAAGCTG�-Actin TGGAATCCTGTGGCATCCTGAAAC TAAAACGCAGCTCAGTAACAGTCCG

Syed et al.

3430 iai.asm.org September 2015 Volume 83 Number 9Infection and Immunity

on March 2, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

cytes with various cell-permeable caspase inhibitors, followed bystimulation with WT S. aureus supernatant. Inhibition of caspase1 (YVAD), caspase 3 (DEVD), caspase 5 (WEHD), caspase 6(VEID), caspase 8 (IEHD), or all caspases (ZVAD) did not impactS. aureus-induced lytic cytokine release (Fig. 3C, D, and F). How-ever, identical concentrations of YVAD and ZVAD repressed in-flammasome activation induced by LPS in PBMCs (Fig. 3E). Ele-vated extracellular potassium, another condition that inhibits

NLR family, pyrin domain containing 3 (NLRP3) inflammasomeactivation, did not inhibit S. aureus-mediated cytokine release(data not shown). These data suggest that release of active IL-18 byPSMs is specific to disruption of cell membranes and is not due tospecific activation of the inflammasome.

Phenol-soluble modulins are required for neutrophil infil-tration following epidermal S. aureus exposure. Given thatPSMs induce lysis of and inflammatory cytokine release from ke-

TSB WT

hla::er

m

agr::

tet PSM

PSM Complem

ent

0

50

100

150

200

250

IL-1

8 (p

g/m

l)

***

*

TSB WT

hla::er

m

agr::

tet PSM

PSM Complem

ent

0

20

40

60

80

100

LDH

Rel

ease

(%)

********

****

TSB WT

hla::er

m

agr::

tet PSM

PSM Complem

ent

0

50

100

150

200

IL-1

(pg/

ml)

***

TSB

S. aureu

s

S. lugdunen

sis

S. epiderm

idis

S. sap

rophyti

cus

S. cap

itis

S. warn

eri

S. hominis

S. hae

molyticu

s0

100

200

300

400

IL-1

8 (p

g/m

l)****

****

**** **** ****

**

TSB

S. aureu

s

S. lugdunen

sis

S. epiderm

idis

S. sap

rophyti

cus

S. cap

itis

S. warn

eri

S. hominis

S. hae

molyticu

s0

20

40

60

80

100

LDH

Rel

ease

(%)

**** **** ********

****BA

DC

E

FIG 1 Various staphylococcal species induce lytic release of IL-18 from human keratinocytes. (A and B) Human keratinocytes were exposed to supernatantsfrom several Staphylococcus species and tested for release of IL-18 (A) and LDH (B). (C to E) Human keratinocytes were also exposed to supernatants from severalstrains of S. aureus and tested for the release of IL-18 (C), LDH (D), and IL-1� (E). ****, P 0.0001; **, P 0.01; *, P 0.05; n � 2 in duplicate for each study.The error bars represent standard errors of the mean.

S. aureus PSMs Promote Skin Inflammation

September 2015 Volume 83 Number 9 iai.asm.org 3431Infection and Immunity

on March 2, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

ratinocytes, we hypothesized that they may be required for initia-tion of inflammation in response to cutaneous colonization by S.aureus. We thus challenged mice epicutaneously with the WT or�psm strain of S. aureus. As shown in Fig. 4A, application of WT S.aureus to the surface of the skin resulted in infectious changes,including erythema and ulceration, whereas the �psm strain pro-duced minimal skin changes. Flow cytometry of the colonized skindemonstrated a significant decrease in neutrophil infiltration ofthe skin in the WT compared to the �psm strain. Other cell types,including macrophages, dendritic cells (DC), B-cells, and T cells,were not significantly altered following S. aureus exposure (Fig.4B). Using C57BL/6 IL-18�/� mice, we found that IL-18 was notrequired for S. aureus-induced neutrophil infiltration into theskin (Fig. 4C). Absence of IL-18 enhanced macrophage, CD11b

DC, and CD4 T cell migration, suggesting that keratinocyte re-lease of IL-18 may have repressive effects on chronic inflamma-tion (Fig. 4C). These data suggest that PSMs are required for in-vasive infection when S. aureus is exposed to intact keratinocytelayers via epicutaneous application and that IL-18 is not requiredfor this process.

Others have demonstrated that epicutaneous application of S.aureus results in upregulation of proinflammatory cytokines (31).In order to determine whether PSMs play a role in modulatinginflammatory gene expression in the skin, we performed RT-PCRon skin sections following exposure to the WT or �psm strain. Asshown in Fig. 5A, both the WT and �psm strains were able todramatically upregulate proinflammatory genes within the skin,including alpha interferon (IFN-�), IFN-�, IL-18, IL-1�, tumornecrosis factor alpha (TNF-�), and IL-6, compared to skin ex-posed to PBS. Both were also able to upregulate the antimicrobial

peptide cathelicidin antimicrobial peptide (CAMP), the murineLL-37 ortholog. Chemokines, such as chemokine (C-C motif) li-gand 4 (CCL4), were also upregulated by both strains. Given thatthe skin phenotype following exposure to the WT versus the �psmstrain was so dramatically different, we then chose to examinesystemic cytokine concentrations in mice exposed to both strains.As shown in Fig. 5B and Table 3, the concentrations of inflamma-tory cytokines, including granulocyte colony-stimulating factor(G-CSF), granulocyte-macrophage colony-stimulating factor(GM-CSF), IL-1�, IL-6, and IL-17, in serum are significantly di-minished in �psm strain-exposed compared to WT-exposedmice. Together, these data suggest that PSMs are not required tostimulate inflammatory gene upregulation but are required to ini-tiate release of proinflammatory cytokines into circulation and topermit the subsequent inflammatory response.

DISCUSSION

S. aureus colonization is known to be highly prevalent in theU.S. population and also contributes to chronic skin condi-tions, such as AD. In this paper, we demonstrate that the PSM�family members, including �-toxin, are necessary and suffi-cient for lytic activity on keratinocytes, which results in therelease of inflammatory cytokines, such as IL-18 and IL-1�(Fig. 1 and 2). Additionally, we demonstrate that PSMs, whilenot required for transcriptional upregulation of proinflamma-tory genes within the skin, are required for instigation of skininflammation and systemic cytokine changes in an epicutane-ous colonization model (Fig. 4 and 5).

Several groups have investigated mechanisms by which S. au-reus can elicit an immune response from keratinocytes; however,

PBS

PSM1

PSM2

PSM3

PSM4

PSM1

PSM2

-toxin

0

100

200

300

400

IL-1

8 (p

g/m

l)

*** ***

***

****

*** ****

PBS

PSM1

PSM2

PSM3

PSM4

PSM1

PSM2

-toxin

0

20

40

60

80

100

LDH

Rel

ease

(%)

*

*******

** **

BA

No Treatm

ent

5 µg/m

l

50 µg

/ml

100 µ

g/ml

0

200

400

600

800

IL-1

8 (p

g/m

l)

PSM 1LL-37

*

*

C

FIG 2 S. aureus PSMs are sufficient for lytic release of IL-18 from human keratinocytes. (A and B) Human keratinocytes were exposed to synthetic PSM peptidesand tested for release of IL-18 (A) and LDH (B). (C) PSMs induce release of IL-18 in a dose-dependent mechanism similar to that of LL-37. ****, P 0.0001; ***,P 0.001; **, P 0.01; *, P 0.05; n � 3 in duplicate for each study. The error bars represent standard errors of the mean.

Syed et al.

3432 iai.asm.org September 2015 Volume 83 Number 9Infection and Immunity

on March 2, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

these factors have required the use of surfactants to promotemembrane permeability (11). Our data presented here demon-strate that keratinocyte-mediated inflammation can be stimulatedby S. aureus PSMs alone. Because of their amphipathic nature,

PSMs have surfactant-like properties that allow membrane inter-calation and pore formation, similar to the human cathelicidinLL-37 (Fig. 2C) (21). Interestingly, not all species of staphylococciinduce keratinocyte lysis (Fig. 1). We observed strong lytic abili-

No Treatm

ent

WT

DMSOYVAD

DEVDVEID

IETDZVAD

0

10

20

30

40

IL-1

(pg/

ml)

No Treatm

entWT

DMSOYVAD

DEVDVEID

IETDZVAD

0

500

1000

1500

2000

IL-1

8 (p

g/m

l)

B

C

TSB

WT

Phase Contrast DapiActive Caspase-1

A

20-

50-

WTTSB rIL-18

Mature

D

Pro

ECM

E

No Treatm

ent

WTDMSO

YVADDEVD

VEIDIETD

ZVAD0

20

40

60

80

LDH

Rel

ease

(%)

IL-1

β (

pg/m

l)

LPS

LPS+YVAD

LPS+ZVAD0

500

1000

1500

2000

****

**

No Treatm

ent

F

FIG 3 The release of mature IL-18 from human keratinocytes in response to S. aureus is independent of caspase 1 function. (A) Human keratinocytes were exposedto TSB or WT S. aureus supernatants for 1 h, followed by addition of a fluorescence-labeled peptide that binds active caspase 1 and visualized by fluorescencemicroscopy (n � 3). (B) Representative Western blotting for full-length, or “pro,” form and active, cleaved IL-18 released into the extracellular media (ECM)from keratinocytes exposed to TSB or WT supernatants. Recombinant active IL-18 (rIL-18) was run as a positive control. (C, D, and F) Dimethyl sulfoxide(DMSO) or caspase inhibitors (10 mM) were added to human keratinocytes prior to treatment with S. aureus supernatants and then testing for the release ofIL-18 (n � 3) (C), IL-1� (n � 3) (D), and LDH (n � 1) (F). (E) Human PBMCs were incubated with 10 mM YVAD or ZVAD for 30 min prior to overnightincubation with 1 mg/ml LPS. Released IL-1� was detected via ELISA. The error bars represent standard errors of the mean. **, P 0.01; ****, P 0.0001.

S. aureus PSMs Promote Skin Inflammation

September 2015 Volume 83 Number 9 iai.asm.org 3433Infection and Immunity

on March 2, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

ties in species known to contain PSMs, including S. aureus, S.epidermidis, and S. lugdunensis, but there has been no study, to ourknowledge, investigating the presence of PSMs in other staphylo-coccal species. Further investigation of S. hominis, which is able toinduce release of IL-18 without inducing cell lysis, will be of inter-est. Additionally, the PSM� peptides were able to induce the re-lease of IL-18 from human keratinocytes without causing a releaseof LDH (Fig. 2). It is possible that the PSM� peptides are able toactivate a receptor-mediated cytokine release or possibly triggerslow, nonlytic inflammasome activation.

Keratinocyte activation and release of IL-18 and IL-1� by caspase

1 as part of an active inflammasome have been reported (37). Addi-tionally, an inflammasome-independent role for caspase 1 in apop-tosis of keratinocytes has been described (38). Interestingly, in thepresence of S. aureus supernatants, we detected activation of caspase1, but this activation is not required for IL-18 release. This suggeststhat PSMs are promoting cytokine release via lysis (Fig. 3) and thatother enzymes, such as cathepsin activation or granzyme B, may beimportant for IL-18 activation in keratinocytes (39). This PSM-in-duced IL-18 release may resolve the contradiction between the role ofincreased IL-18 signaling in atopic dermatitis and recent data thatdescribe repressed inflammasome activity in AD (40, 41). Chronic

A PBS WT ∆psm

Neutro

phils

Macro

phages

CD11b- D

Cs

CD8+ T-ce

lls

CD4+ T-ce

lls

B-cells

0

200

400

600

Cel

ls/5

0,00

0 ev

ents

W Tpsm

PBS*

CD11b+ D

Cs

B

Cel

ls/5

0,00

0ev

ents

Neutro

phils

Macro

phages

CD11b- DC

CD11b+ DC

CD8+T ce

lls

CD4+T ce

lls

CD19+ B

cells

0

100

200

300

400

WTIL-18 KO

****

*** ****C

FIG 4 PSMs are critical for the recruitment of neutrophils into the skin during S. aureus epidermal exposure. (A) Representative photographs of dorsal surfacesfrom mice taken after 7 days of exposure. (B) FACS analysis of leukocytes obtained from skin samples from mice 7 days postinoculation with S. aureus. *, P 0.05;n � 8 for each group. (C) FACS analysis of skin leukocytes from WT or IL-18 knockout (KO) mice exposed (n � 5) to WT S. aureus, as in panel B. Neutrophils,Ly6G ; macrophages, F4/80 CD11b ; dendritic cells, Ly6c CD11c ; T cells, CD3 ; B cells, CD19 . The error bars represent standard errors of the mean. ***,P 0.001; ****, P 0.0001.

IFN IFN IFNIL-18 IL-1 IL-6

IL-1TNF

CAMP

NLRP3CCR1

CCL4CCL5

CXCL10

CXCL13

-200

2040

200

300

400

FC v

s. c

ontr

ol

WTpsm

G-CSF

GM-CSF

IL-1 IL-6IL-17

0

50

100300400500600

3000400050006000

Cyt

okin

e Le

vel (

pg/m

l) WTpsm

**

*****

**

**

BA

FIG 5 Systemic inflammatory response, but not inflammatory gene upregulation, requires S. aureus PSMs. (A) Fold changes (FC) in the cutaneous expressionof the listed genes compared to PBS-exposed mice as calculated by real-time PCR. (B) Concentrations of the indicated cytokines in serum as determined byMilliplex in WT S. aureus-exposed versus S. aureus �psm-exposed mice. **, P 0.01; ***, P 0.001; n � 8 for each group. The error bars represent standarderrors of the mean.

Syed et al.

3434 iai.asm.org September 2015 Volume 83 Number 9Infection and Immunity

on March 2, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

colonization with PSM-producing S. aureus may circumvent theneed for inflammasome-mediated IL-18 release.

In our epicutaneous model of S. aureus exposure, we foundthat absence of IL-18 did not impact neutrophil recruitment butenhanced chronic inflammatory cell infiltration. This is an inter-esting finding, as the actual function of IL-18 in the skin has notbeen systematically investigated. Many studies examining the roleof IL-18 in cutaneous disease report up- or downregulation of thecytokine without reporting the consequence of these changes (42).Our data suggest that following S. aureus exposure, IL-18 mayfunction to modulate the acute versus chronic inflammatory re-sponse. Given that absence of IL-18 promoted a large increase inCD4 T cells in our study, further investigation into the Th1 ver-sus Th2 skewing of these populations is warranted. Additionally,determining whether the effects of IL-18 are different in AD mod-els exposed to S. aureus is an important area for future research.

PSMs produced by S. aureus have been shown to be criticaldeterminants of pathogenesis in murine models of abscess andbacteremia (19). They are recognized by the human formyl pep-tide receptor 2, which stimulates neutrophil chemotaxis (24). Notonly can PSMs recruit neutrophils, but they have been shown tointercalate into cell membranes, resulting in lysis and death (19).In our in vivo model, transcriptional changes of cytokines in thedermis in our mice did not differ between mice exposed to WT S.aureus and mice exposed to S. aureus �psm (Fig. 5A). This likelyreflects intact Toll-like receptor (TLR) ligands, such as pepti-doglycan, present on each strain. Keratinocytes express TLR2,which results in robust inflammatory gene upregulation after ex-

posure to peptidoglycan (43). Thus, it is not surprising to seerobust gene expression changes in the skin after exposure to theWT and �psm strains. Importantly, however, while a robust sys-temic inflammatory response was noted with WT S. aureus, thecirculating concentrations of inflammatory cytokines were dra-matically and significantly reduced after exposure to S. aureus�psm (Fig. 5B). The large difference in G-CSF agrees with previ-ously reported effects of one of the PSMs, �-toxin, on mast celldegranulation and G-CSF release (44). Differences in circulatingIL-6 and IL-1�, both of which are released from keratinocytes,were also noted (Fig. 3) (37, 45). Systemic levels of IL-18 were notaltered after S. aureus exposure (Table 3), but this does not ruleout important localized effects, as other cytokines important inAD have been noted to be increased in the skin but not in serum(46). Taken together with our in vitro data, which demonstratePSM-mediated release of IL-18 and IL-1� from keratinocytes (Fig.1C and E and 5B), these results suggest that PSM-mediated kera-tinocyte lysis may be a required factor to convert S. aureus colo-nization into infection.

In summary, we have described a novel role for staphylococcalPSMs, which induce keratinocyte lysis and release of inflamma-tory cytokines. This translates into a role of PSMs in infection andthe systemic inflammatory response in vivo. We propose thatPSMs are important for initiating pathogenesis after colonizationand may serve as important targets for treating infection or mod-ulating inflammatory cutaneous disease.

ACKNOWLEDGMENTS

We thank the laboratory of Johann Gudjonsson at the University of Mich-igan for providing cells and protocols and allowing us the use of theirequipment. We also thank the members of the Kahlenberg laboratory atthe University of Michigan, as well as Matthew Brown in the Boles labo-ratory at the University of Iowa, for insightful conversations.

This work was funded by the National Institute of Arthritis and Mus-culoskeletal and Skin Diseases of the National Institutes of Health underaward number K08AR063668 to J.M.K., NIH grant NIAID AI081748 toB.R.B., and American Heart Association Fellowship 13PRE13810001 toA.K.S.

REFERENCES1. Klevens RM, Morrison MA, Nadle J, Petit S, Gershman K, Ray S,

Harrison LH, Lynfield R, Dumyati G, Townes JM, Craig AS, Zell ER,Fosheim GE, McDougal LK, Carey RB, Fridkin SK, Active BacterialCore Surveillance (ABCs) Investigators MRSA. 2007. Invasive methicil-lin-resistant Staphylococcus aureus infections in the United States. JAMA298:1763–1771. http://dx.doi.org/10.1001/jama.298.15.1763.

2. Kuehnert MJ, Kruszon-Moran D, Hill HA, McQuillan G, McAllisterSK, Fosheim G, McDougal LK, Chaitram J, Jensen B, Fridkin SK,Killgore G, Tenover FC. 2006. Prevalence of Staphylococcus aureus nasalcolonization in the United States, 2001–2002. J Infect Dis 193:172–179.http://dx.doi.org/10.1086/499632.

3. Sievert DM, Ricks P, Edwards JR, Schneider A, Patel J, Srinivasan A,Kallen A, Limbago B, Fridkin S, National Healthcare Safety Network(NHSN) Team and Participating Facilities NHSN. 2013. Antimicrobial-resistant pathogens associated with healthcare-associated infections: sum-mary of data reported to the National Healthcare Safety Network at theCenters for Disease Control and Prevention, 2009 –2010. Infect ControlHosp Epidemiol 34:1–14. http://dx.doi.org/10.1086/668770.

4. De Benedetto A, Agnihothri R, McGirt LY, Bankova LG, Beck LA. 2009.Atopic dermatitis: a disease caused by innate immune defects? J InvestigDermatol 129:14 –30. http://dx.doi.org/10.1038/jid.2008.259.

5. Nutten S. 2015. Atopic dermatitis: global epidemiology and risk fac-tors. Ann Nutr Metab 66(Suppl 1):S8 –S16. http://dx.doi.org/10.1159/000370220.

6. Foster TJ, Hook M. 1998. Surface protein adhesins of Staphylococcus

TABLE 3 Cytokine concentrations from mice exposed to WT S. aureus,S. aureus �psm, or vehicle control (PBS)

Cytokine

Concn (pg/ml) after exposure to:

Significant differencebetween WT S.aureus- and S. aureus�psm-exposed mice(P value)

WTS. aureus

S. aureus�psm PBS

G-CSF 3443.4 287.9 434.1 Yes (0.0011)GM-CSF 34.5 12.1 14.8 Yes (0.00540)IFN� 2.5 2.0 2.1 No (0.2000)IL-1� 249.6 266.4 275.4 No (0.0827)IL-1� 16.6 7.9 22.7 Yes (0.0009)IL-2 3.6 2.7 3.7 Yes (0.0256)IL-4 3.0 3.0 3.4 No (1.0000)IL-5 8.4 5.6 17.8 No (0.2887)IL-6 71.6 9.0 23.5 Yes (0.0070)IL-7 3.4 1.0 18.5 Yes (0.0002)IL-10 3.8 2.1 3.0 Yes (0.0020)IL-12p40 4.9 3.7 3.5 No (0.2822)IL-12p70 10.2 3.7 13.0 Yes (0.00480)IL-13 84.7 45.7 58.7 Yes (0.0047)IL-15 6.8 2.5 2.5 Yes (0.0256)IL-17 11.7 3.0 3.3 Yes (0.00280)IL-18 193.8 201.1 190.9 No (0.769)IP-10 142.7 101.9 80.4 Yes (0.0379)KC 116.3 81.5 104.1 No (0.3754)MCP-1 63.9 25.4 24.4 No (0.0692)MIP-1a 39.5 13.6 19.5 Yes (0.0006)MIP-1b 18.7 9.0 13.1 No (0.2124)MIP-2 88.0 75.1 60.4 No (0.3958)RANTES 14.9 9.4 7.1 No (0.2247)TNF-� 5.2 3.4 3.6 No (0.0816)

S. aureus PSMs Promote Skin Inflammation

September 2015 Volume 83 Number 9 iai.asm.org 3435Infection and Immunity

on March 2, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

aureus. Trends Microbiol 6:484 – 488. http://dx.doi.org/10.1016/S0966-842X(98)01400-0.

7. Travers JB, Norris DA, Leung DY. 2001. The keratinocyte as a target forstaphylococcal bacterial toxins. J Investig Dermatol Symp Proc 6:225–230.http://dx.doi.org/10.1046/j.0022-202x.2001.00045.x.

8. Sasaki T, Kano R, Sato H, Nakamura Y, Watanabe S, Hasegawa A. 2003.Effects of staphylococci on cytokine production from human keratino-cytes. Br J Dermatol 148:46 –50. http://dx.doi.org/10.1046/j.1365-2133.2003.05017.x.

9. Travers JB, Kozman A, Mousdicas N, Saha C, Landis M, Al-Hassani M,Yao W, Yao Y, Hyatt A-M, Sheehan MP, Haggstrom AN, Kaplan MH.2010. Infected atopic dermatitis lesions contain pharmacologic amountsof lipoteichoic acid. J Allergy Clin Immunol 125:146 –152.e1–2. http://dx.doi.org/10.1016/j.jaci.2009.09.052.

10. Yao Y, Kozman A, Al-Hassani M, Saha CK, Yi Q, Yao W, Mousdicas N,Kaplan MH, Travers JB. 2010. Identification of staphylococcal protein Ain infected atopic dermatitis lesions. J Investig Dermatol 130:2502–2504.http://dx.doi.org/10.1038/jid.2010.154.

11. Terada M, Tsutsui H, Imai Y, Yasuda K, Mizutani H, Yamanishi K,Kubo M, Matsui K, Sano H, Nakanishi K. 2006. Contribution of IL-18to atopic-dermatitis-like skin inflammation induced by Staphylococcusaureus product in mice. Proc Natl Acad Sci U S A 103:8816 – 8821. http://dx.doi.org/10.1073/pnas.0602900103.

12. Ommori R, Ouji N, Mizuno F, Kita E, Ikada Y, Asada H. 2013. Selectiveinduction of antimicrobial peptides from keratinocytes by staphylococcalbacteria. Microb Pathog 56:35–39. http://dx.doi.org/10.1016/j.micpath.2012.11.005.

13. Higashi N, Gesser B, Kawana S, Thestrup-Pedersen K. 2001. Expressionof IL-18 mRNA and secretion of IL-18 are reduced in monocytes frompatients with atopic dermatitis. J Allergy Clin Immunol 108:607– 614.http://dx.doi.org/10.1067/mai.2001.118601.

14. Yamanaka K, Tanaka M, Tsutsui H, Kupper TS, Asahi K, Okamura H,Nakanishi K, Suzuki M, Kayagaki N, Black RA, Miller DK, NakashimaK, Shimizu M, Mizutani H. 2000. Skin-specific caspase-1-transgenicmice show cutaneous apoptosis and pre-endotoxin shock condition with ahigh serum level of IL-18. J Immunol 165:997–1003. http://dx.doi.org/10.4049/jimmunol.165.2.997.

15. Fantuzzi G, Puren AJ, Harding MW, Livingston DJ, Dinarello CA.1998. Interleukin-18 regulation of interferon gamma production and cellproliferation as shown in interleukin-1beta-converting enzyme (caspase-1)-deficient mice. Blood 91:2118 –2125.

16. Lamkanfi M, Dixit VM. 2012. Inflammasomes and their roles in healthand disease. Annu Rev Cell Dev Biol 28:137–161. http://dx.doi.org/10.1146/annurev-cellbio-101011-155745.

17. Thornberry NA, Bull HG, Calaycay JR, Chapman KT, Howard AD,Kostura MJ, Miller DK, Molineaux SM, Weidner JR, Aunins J, EllistonKO, Ayala JM, Casano FJ, Chin J, Ding J-F, Egger LA, Gaffney EP,Limjuco G, Palyha OC, Raju SM, Rolando AM, Salley JP, Yamin TT,Lee TD, Shivey JE, MacCross M, Mumford RA, Schmidt JA, Tocci MJ.1992. A novel heterodimeric cysteine protease is required for interleu-kin-1� processing in monocytes. Nature 356:768 –774. http://dx.doi.org/10.1038/356768a0.

18. Sahoo M, Ceballos-Olvera I, del Barrio L, Re F. 2011. Role of theinflammasome, IL-1�, and IL-18 in bacterial infections. ScientificWorld-Journal 11:2037–2050. http://dx.doi.org/10.1100/2011/212680.

19. Wang R, Braughton KR, Kretschmer D, Bach T-HL, Queck SY, Li M,Kennedy AD, Dorward DW, Klebanoff SJ, Peschel A, DeLeo FR, OttoM. 2007. Identification of novel cytolytic peptides as key virulence deter-minants for community-associated MRSA. Nat Med 13:1510 –1514. http://dx.doi.org/10.1038/nm1656.

20. Tsompanidou E, Denham EL, Becher D, de Jong A, Buist G, van OostenM, Manson WL, Back JW, van Dijl JM, Dreisbach A. 2013. Distinct rolesof phenol-soluble modulins in spreading of Staphylococcus aureus on wetsurfaces. Appl Environ Microbiol 79:886 – 895. http://dx.doi.org/10.1128/AEM.03157-12.

21. Tsompanidou E, Sibbald MJJB, Chlebowicz MA, Dreisbach A, Back JW,van Dijl JM, Buist G, Denham EL. 2011. Requirement of the agr locus forcolony spreading of Staphylococcus aureus. J Bacteriol 193:1267–1272.http://dx.doi.org/10.1128/JB.01276-10.

22. Periasamy S, Joo H-S, Duong AC, Bach T-HL, Tan VY, Chatterjee SS,Cheung GYC, Otto M. 2012. How Staphylococcus aureus biofilms de-velop their characteristic structure. Proc Natl Acad Sci U S A 109:1281–1286. http://dx.doi.org/10.1073/pnas.1115006109.

23. Schwartz K, Syed AK, Stephenson RE, Rickard AH, Boles BR. 2012.Functional amyloids composed of phenol soluble modulins stabilizeStaphylococcus aureus biofilms. PLoS Pathog 8:e1002744. http://dx.doi.org/10.1371/journal.ppat.1002744.

24. Kretschmer D, Gleske A-K, Rautenberg M, Wang R, Köberle M, BohnE, Schöneberg T, Rabiet M-J, Boulay F, Klebanoff SJ, van Kessel KA,van Strijp JA, Otto M, Peschel A. 2010. Human formyl peptide receptor2 senses highly pathogenic Staphylococcus aureus. Cell Host Microbe7:463– 473. http://dx.doi.org/10.1016/j.chom.2010.05.012.

25. Surewaard BGJ, Nijland R, Spaan AN, Kruijtzer JAW, de Haas CJC, vanStrijp JAG. 2012. Inactivation of staphylococcal phenol soluble modulinsby serum lipoprotein particles. PLoS Pathog 8:e1002606. http://dx.doi.org/10.1371/journal.ppat.1002606.

26. Cogen AL, Yamasaki K, Sanchez KM, Dorschner RA, Lai Y, MacLeodDT, Torpey JW, Otto M, Nizet V, Kim JE, Gallo RL. 2010. Selectiveantimicrobial action is provided by phenol-soluble modulins derivedfrom Staphylococcus epidermidis, a normal resident of the skin. J InvestDermatol 130:192–200. http://dx.doi.org/10.1038/jid.2009.243.

27. Joo HS, Cheung GYC, Otto M. 2011. Antimicrobial activity of commu-nity-associated methicillin-resistant Staphylococcus aureus is caused byphenol-soluble modulin derivatives. J Biol Chem 286:8933– 8940. http://dx.doi.org/10.1074/jbc.M111.221382.

28. Gonzalez DJ, Okumura CY, Hollands A, Kersten R, Akong-Moore K,Pence MA, Malone CL, Derieux J, Moore BS, Horswill AR, Dixon JE,Dorrestein PC, Nizet V. 2012. Novel phenol-soluble modulin derivativesin community-associated methicillin-resistant Staphylococcus aureusidentified through imaging mass spectrometry. J Biol Chem 287:13889 –13898. http://dx.doi.org/10.1074/jbc.M112.349860.

29. Mehlin C, Headley CM, Klebanoff SJ. 1999. An inflammatory polypep-tide complex from Staphylococcus epidermidis: isolation and character-ization. J Exp Med 189:907–918. http://dx.doi.org/10.1084/jem.189.6.907.

30. Donvito B, Etienne J, Denoroy L, Greenland T, Benito Y, VandeneschF. 1997. Synergistic hemolytic activity of Staphylococcus lugdunensis ismediated by three peptides encoded by a non-agr genetic locus. InfectImmun 65:95–100.

31. Wanke I, Skabytska Y, Kraft B, Peschel A, Biedermann T, Schittek B.2013. Staphylococcus aureus skin colonization is promoted by barrierdisruption and leads to local inflammation. Exp Dermatol 22:153–155.http://dx.doi.org/10.1111/exd.12083.

32. Boles BR, Thoendel M, Roth AJ, Horswill AR. 2010. Identification ofgenes involved in polysaccharide-independent Staphylococcus aureusbiofilm formation. PLoS One 5:e10146. http://dx.doi.org/10.1371/journal.pone.0010146.

33. Schwartz K, Sekedat MD, Syed AK, O’Hara B, Payne DE, Lamb A, BolesBR. 2014. The AgrD N-terminal leader peptide of Staphylococcus aureushas cytolytic and amyloidogenic properties. Infect Immun 82:3837–3844.http://dx.doi.org/10.1128/IAI.02111-14.

34. Kebaier C, Chamberland RR, Allen IC, Gao X, Broglie PM, Hall JD,Jania C, Doerschuk CM, Tilley SL, Duncan JA. 2012. Staphylococcusaureus �-hemolysin mediates virulence in a murine model of severe pneu-monia through activation of the NLRP3 inflammasome. J Infect Dis 205:807– 817. http://dx.doi.org/10.1093/infdis/jir846.

35. Kahlenberg JM, Kaplan MJ. 2013. Little peptide, big effects: the role ofLL-37 in inflammation and autoimmune disease. J Immunol 191:4895–4901. http://dx.doi.org/10.4049/jimmunol.1302005.

36. Muñoz-Planillo R, Franchi L, Miller LS, Núñez G. 2009. A critical rolefor hemolysins and bacterial lipoproteins in Staphylococcus aureus-induced activation of the Nlrp3 inflammasome. J Immunol 183:3942–3948. http://dx.doi.org/10.4049/jimmunol.0900729.

37. Dai X, Sayama K, Tohyama M, Shirakata Y, Hanakawa Y, TokumaruS, Yang L, Hirakawa S, Hashimoto K. 2011. Mite allergen is a dangersignal for the skin via activation of inflammasome in keratinocytes. JAllergy Clin Immunol 127:806 – 814.e1– 4. http://dx.doi.org/10.1016/j.jaci.2010.12.006.

38. Sollberger G, Strittmatter GE, Grossi S, Garstkiewicz M, Auf dem KellerU, French LE, Beer H-D. 2015. Caspase-1 activity is required for UVB-induced apoptosis of human keratinocytes. J Investig Dermatol 135:1395–1404. http://dx.doi.org/10.1038/jid.2014.551.

39. Akeda T, Yamanaka K, Tsuda K, Omoto Y, Gabazza EC, Mizutani H.2014. CD8 T cell granzyme B activates keratinocyte endogenous IL-18.Arch Dermatol Res 306:125–130. http://dx.doi.org/10.1007/s00403-013-1382-1.

40. Inoue Y, Aihara M, Kirino M, Harada I, Komori-Yamaguchi J, Yama-

Syed et al.

3436 iai.asm.org September 2015 Volume 83 Number 9Infection and Immunity

on March 2, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

guchi Y, Nagashima Y, Ikezawa Z. 2011. Interleukin-18 is elevated in thehorny layer in patients with atopic dermatitis and is associated with Staph-ylococcus aureus colonization. Br J Dermatol 164:560 –567. http://dx.doi.org/10.1111/j.1365-2133.2010.10145.x.

41. Niebuhr M, Baumert K, Heratizadeh A, Satzger I, Werfel T. 2014.Impaired NLRP3 inflammasome expression and function in atopic der-matitis due to Th2 milieu. Allergy 69:1058 –1067. http://dx.doi.org/10.1111/all.12428.

42. Puxeddu I, Italiani P, Giungato P, Pratesi F, Panza F, Bartaloni D,Rocchi V, Del Corso I, Boraschi D, Migliorini P. 2013. Free IL-18 andIL-33 cytokines in chronic spontaneous urticaria. Cytokine 61:741–743.http://dx.doi.org/10.1016/j.cyto.2013.01.015.

43. Yoshimura A, Lien E, Ingalls RR, Tuomanen E, Dziarski R, GolenbockD. 1999. Cutting edge: recognition of gram-positive bacterial cell wallcomponents by the innate immune system occurs via Toll-like receptor 2.J Immunol 163:1–5.

44. Nakamura Y, Oscherwitz J, Cease KB, Chan SM, Muñoz-Planillo R,Hasegawa M, Villaruz AE, Cheung GYC, McGavin MJ, Travers JB, Otto

M, Inohara N, Núñez G. 2013. Staphylococcus �-toxin induces allergicskin disease by activating mast cells. Nature 503:397– 401. http://dx.doi.org/10.1038/nature12655.

45. Wozniacka A, Lesiak A, Boncela J, Smolarczyk K, McCauliffe DP,Sysa-Jedrzejowska A. 2008. The influence of antimalarial treatment onIL-1beta, IL-6 and TNF-alpha mRNA expression on UVB-irradiated skinin systemic lupus erythematosus. Br J Dermatol 159:1124 –1130. http://dx.doi.org/10.1111/j.1365-2133.2008.08804.x.

46. Chen Y, Lind Enoksson S, Johansson C, Karlsson MA, Lundeberg L,Nilsson G, Scheynius A, Karlsson MCI. 2011. The expression of BAFF,APRIL and TWEAK is altered in eczema skin but not in the circulation ofatopic and seborrheic eczema patients. PLoS One 6:e22202. http://dx.doi.org/10.1371/journal.pone.0022202.

47. Kiedrowski MR, Kavanaugh JS, Malone CL, Mootz JM, Voyich JM,Smeltzer MS, Bayles KW, Horswill AR. 2011. Nuclease modulates bio-film formation in community-associated methicillin-resistant Staphylo-coccus aureus. PLoS One 6:e26714. http://dx.doi.org/10.1371/journal.pone.0026714.

S. aureus PSMs Promote Skin Inflammation

September 2015 Volume 83 Number 9 iai.asm.org 3437Infection and Immunity

on March 2, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

Erratum for Syed et al., Staphylococcus aureus Phenol-SolubleModulins Stimulate the Release of Proinflammatory Cytokines fromKeratinocytes and Are Required for Induction of Skin Inflammation

Adnan K. Syed,a,b Tamra J. Reed,c Kaitlyn L. Clark,c Blaise R. Boles,b J. Michelle Kahlenbergc

Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan, USAa; Department of Microbiology, Roy J. and Lucille A.Carver College of Medicine, University of Iowa, Iowa City, Iowa, USAb; Department of Internal Medicine, Division of Rheumatology, University of Michigan, Ann Arbor,Michigan, USAc

Volume 83, no. 9, p. 3428 –3437, 2015. Page 3428: The article title should read as given above.

Page 3428: The article citation in the footnote block should read as given below.

Citation Syed AK, Reed TJ, Clark KL, Boles BR, Kahlenberg JM. 2015. Staphylococcus aureus phenol-soluble modulins stimulate therelease of proinflammatory cytokines from keratinocytes and are required for induction of skin inflammation. Infect Immun83:3428 –3437. doi:10.1128/IAI.00401-15.

Citation Syed AK, Reed TJ, Clark KL, Boles BR, Kahlenberg JM. 2015. Erratum forSyed et al., Staphylococcus aureus phenol-soluble modulins stimulate the releaseof proinflammatory cytokines from keratinocytes and are required for induction ofskin inflammation. Infect Immun 83:4450. doi:10.1128/IAI.01087-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

ERRATUM

4450 iai.asm.org November 2015 Volume 83 Number 11Infection and Immunity