Polymorphonuclear leukocytes mediateStaphylococcus aureus Panton-Valentine leukocidin-induced lung inflammation and injuryBinh An Diepa,1, Liana Chana, Pierre Tattevina,b,c, Osamu Kajikawad,e, Thomas R. Martind,e, Li Basuinoa, Thuy T. Maia,Helene Marbacha, Kevin R. Braughtonf, Adeline R. Whitneyf, Donald J. Gardnerg, Xuemo Fanh, Ching W. Tsengi,j,George Y. Liui,j, Cedric Badiouk,l, Jerome Etiennek,l,m, Gerard Linak,l,m, Michael A. Matthayn, Frank R. DeLeof,and Henry F. Chambersa,1

aDivision of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94110; bMaladies Infectieuses et Réanimation Médicale,CHU Pontchaillou, 35033 Rennes, France; cInstitut National de la Santé et de la Recherche Médicale U835, Université Rennes I, 35033 Rennes, France; dMedicalResearch Service of the Veterans Affairs and Puget Sound Health Care System and eDivision of Pulmonary and Critical Care Medicine, Department ofMedicine, University of Washington School of Medicine, Seattle, WA 98108; fLaboratory of Human Bacterial Pathogenesis and gVeterinary Branch, RockyMountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840; hDepartment of Pathologyand Laboratory Medicine and iDepartment of Pediatrics and the Immunobiology Research Institute, Cedars-Sinai Medical Center and jUniversity of California,Los Angeles, CA 90048; kFaculté de Médecine Laennec, Université Lyon 1, 69008 Lyon, France; lInstitut National de la Santé et de la Recherche Médicale U851,Centre National de Référence des Staphylocoques, 69008 Lyon, France; mHospices Civils de Lyon, Centre de Biologie Est, 69500 Lyon, France; and nDepartmentof Medicine and Anesthesia, Cardiovascular Research Institute, University of California, San Francisco, CA 94143

Edited* by Richard P. Novick, NYU School of Medicine, New York, NY, and approved February 10, 2010 (received for review November 4, 2009)

Community-associated methicillin-resistant Staphylococcus aureus(CA-MRSA) is epidemic in the United States, even rivaling HIV/AIDSin its public health impact. The pandemic clone USA300, like otherCA-MRSA strains, expresses Panton-Valentine leukocidin (PVL), apore-forming toxin that targets polymorphonuclear leukocytes(PMNs). PVL is thought to play a key role in the pathogenesis ofnecrotizing pneumonia, but data from rodent infection models areinconclusive. Rodent PMNs are less susceptible than human PMNs toPVL-induced cytolysis, whereas rabbit PMNs, like those of humans,are highly susceptible to PVL-induced cytolysis. This difference intarget cell susceptibility could affect results of experimental models.Therefore, we developed a rabbit model of necrotizing pneumoniato compare the virulence of a USA300 wild-type strain with that ofisogenic PVL-deletion mutant and -complemented strains. PVLenhanced the capacity of USA300 to cause severe lung necrosis,pulmonary edema, alveolar hemorrhage, hemoptysis, and death,hallmark clinical features of fatal human necrotizing pneumonia.Purified PVL instilled directly into the lung caused lung inflamma-tion and injury by recruiting and lysing PMNs, which damage thelung by releasing cytotoxic granule contents. These findings provideinsights into the mechanism of PVL-induced lung injury and inflam-mation and demonstrate the utility of the rabbit for studying PVL-mediated pathogenesis.

MRSA | USA300 | virulence | pneumonia | rabbit infection model

Community-associated methicillin-resistant Staphylococcus aur-eus (CA-MRSA), especially the pandemic USA300 clone, has

been associated with severe infections and high mortality rates,particularly in patients with fulminant necrotizing pneumonia (1–5).USA300, like other CA-MRSA strains, produces Panton-Valentineleukocidin (PVL), amemberof the family of bicomponent β-channelpore-forming toxins. PVL targets phagocytic leukocytes, especiallypolymorphonuclear leukocytes (PMNs), the first line of defenseagainst S. aureus infections (6–9). Compelling epidemiological datapoint to PVL as an important virulence factor in S. aureus necroti-zing infections, but experimental data in rodent models are incon-clusive (10–18).In contrast to mouse and nonhuman primate PMNs, which are

relatively resistant to PVL in vitro, human and rabbit PMNs aresusceptible to its cytotoxic effects (6–9). This difference in targetcell susceptibility could affect results obtained with different animalspecies (8, 9). The susceptibility of host PMNs to PVL may be animportant consideration when selecting an appropriate animalspecies to model its role in pathogenesis. Therefore, if PMNs are

the relevant target in vivo, the rabbit might be the ideal model totest whether PVL contributes to the pathogenesis of necrotizingpneumonia.Despite the strong clinical association of PVL with severe

necrotizing infections, mechanisms by which this toxin inducestissue necrosis are not known. One possibility is that PVL-induced PMN lysis results in impaired host defenses, interferingwith clearance of organisms from the site of infection andallowing unchecked bacterial growth and expression of othertissue-damaging exotoxins (e.g., α-hemolysin). Another possi-bility is that PVL itself directly or indirectly causes tissue injury.In vitro, PVL activates PMNs to release potent proinflammatorymediators (IL-8 and leukotriene-B4) and granule enzymes(β-glucuronidase, hydrolase, and lysozyme) and to producereactive oxygen metabolites that may cause tissue injury (7, 19–22). Understanding the mechanisms by which PVL promotespathogenesis in animal models should clarify its role in humaninfections and may provide a basis for development of newtherapeutic interventions. To these ends, we developed a rabbitmodel of necrotizing pneumonia to study specific mechanisms ofPVL-induced acute lung injury.

Results and DiscussionPVL Is a Potent Cytolytic Toxin for Human and Rabbit PMNs.As a steptoward determining whether the rabbit would be a suitable ani-mal model in which to test a potential role of PVL in USA300necrotizing pneumonia, we used previously described methods(12, 23–25) to evaluate the ability of purified PVL to formmembrane pores or lyse human and rabbit PMNs. Rabbit andhuman PMNs were highly susceptible to purified PVL (LukS-PV+ LukF-PV), as measured by membrane pore formation andcytolysis in vitro (Fig. 1 A and B). PVL from culture supernatantsof PVL-expressing SF8300, a USA300 clinical strain, but notfrom an isogenic lukS/F-PV–negative mutant (Δpvl) strain, also

Author contributions: B.A.D. andH.F.C. designed research; B.A.D., L.C., P.T., O.K., L.B., T.T.M.,H.M., K.R.B., A.R.W., C.W.T., and C.B. performed research; B.A.D., O.K., T.R.M., J.E., G.L., andF.R.D. contributed new reagents/analytic tools; B.A.D., P.T., D.J.G., X.F., G.Y.L., G.L., M.A.M.,F.R.D., and H.F.C. analyzed data; and B.A.D. and H.F.C. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence may be addressed. E-mail: bdiep@medsfgh.ucsf.edu orhchambers@medsfgh.ucsf.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0912403107/DCSupplemental.

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caused pores in plasma membranes of human and rabbit PMNsat low concentration (Fig. 1C).

PVL Is a Virulence Determinant in a Rabbit Pneumonia Model. In adose-escalation study, endotracheal instillation of 1 × 108 and5 × 108 cfu of SF8300 in outbred rabbits resulted in no mortality,whereas treatment with 1 × 109, 5 × 109, and 10 × 109 cfu caused40–100% mortality. The higher inoculum, which on a per weight

basis corresponds to 10-fold fewer bacteria than used in mousepneumonia models (11, 13), was selected to compare the viru-lence of SF8300 wild-type and Δpvl strains.The wild-type strain caused greater, dose-dependent mor-

tality (Fig. 2A) and more frequent hemoptysis (35/45 vs. 18/43,P < 0.001) than the Δpvl strain. With both the wild-type strainand Δpvl mutants, mortality was associated with high titers ofbacteria in the lungs (Fig. 2B). Survival was associated withclearance of organisms, evidenced by 104-fold cfu fewer in thelungs of rabbits killed at 48 h as compared with those that diedfrom infection (open vs. filled symbols). The significantly highernumbers of bacteria recovered from the lungs of rabbitsinfected with the wild-type strain compared with the Δpvl strain(Fig. 2B) could reflect the rapidly lethal course of infectioncaused by the wild-type strain that prevented host clearance ofthese bacteria, a hypothesis explored more fully in the time-course experiments described later.Lung histopathology showed that wild-type strains (12 lungs

scored) and Δpvl strains (11 lungs scored) induced lung injury(Table S1), a finding consistent with the capacity of PVL-negative S. aureus to elaborate other virulence determinants, suchas staphylococcal protein A and α-hemolysin, which also contributeto acute lung injury (13, 26, 27). However, compared with the Δpvlstrain, the wild-type strain caused significantly more extensivenecrosis with disruption of pulmonary architecture, hemorrhagicinfiltration, exudate/fibrin deposition, alveolar and interstitialedema, and PMN infiltration and destruction (Table S1). Pulmo-nary edema was more severe in rabbits infected with the wild-typestrain than in animals infected with theΔpvl strain, as evidenced bysignificantly greater lung wet weight to body weight (LW/BW)ratios (Fig. 2C). Severe pulmonary edema (i.e., LW/BW ratio>12),which was accompanied by profound hypoxemia [mean arterialpartial pressure of oxygen (PO2), 33 on room air], respiratory failure(mean arterial PCO2, 78), and both respiratory and metabolicacidosis (mean arterial pH 7.14 and lactate 15.0 mmol/L) wasobserved more frequently in rabbits infected with the wild-typestrain than in those infected with the Δpvl strain (Fig. 2C).

Restoration of PVL Production in the Δpvl Strain Restored BacterialVirulence. The Δpvl strain was significantly less virulent thaneither the wild-type or PVL-complemented Δpvl (compΔpvl)strains, as determined by animal mortality, cfu, and LW/BWratios (Fig. 2 D–F). Levels of IL-8, but not monocyte chemo-tactic protein 1 (MCP-1), were significantly higher in animalsinfected with wild-type and compΔpvl strains than in animalsinfected with the Δpvl strain (Fig. 2 G and H). PVL-inducedproduction of IL-8 in the lung reported here is consistent withprevious observations that PVL stimulates PMNs to produceIL-8 in vitro (21, 28).

PVL Is Produced in Toxic Amounts in the Lung. PVL expression in therabbit lung was measured by ELISA as previously described (29,30). LukS-PV was produced in 12–18 μg per lung in rabbitsinfected with 6 × 109 cfu of either wild-type or compΔpvl strains;no LukS-PV was detected in the lungs of rabbits infected withthe Δpvl strain (Fig. 2I). PVL was not detected in the lungs ofrabbits infected with 1.5 × 109 cfu of the wild-type strain, indi-cating that a high bacterial burden in the rabbit lung is requiredfor PVL to be produced in amounts that are comparable to thoseachieved during human skin infections (31). It is of interestthat inocula for the wild-type and compΔpvl strains contained0.1–0.2 μg of LukS-PV. Therefore, de novo PVL productionduring infection, not preformed toxin, was responsible for theincreased virulence of wild-type and compΔpvl strains.

Time-Course of PVL-Induced Acute Lung Injury. Although PVL isstrongly associated with necrotizing pneumonia and high mor-tality (Fig. 2), it is unclear whether PVL promotes bacterial

Fig. 1. Pore-forming and cytolytic activities of PVL toward human andrabbit PMNs. (A) PMN pore formation [percent ethidium bromide (EtBr)-positive cells] and (B) PMN lysis [percent lactate dehydrogenase (LDH)release] assays using LukS-PV and LukF-PV purified from culture super-natants of a PVL-producing USA300 strain as described in SI Text. Results in Aand B are mean of independent experiments with six human and six rabbitblood donors. (C) Pore formation caused by 1:2,000 dilutions of 8-h CCYculture supernatants of USA300 wild-type strain, Δpvl mutant, andcompΔpvl strain as indicated. Results in C are mean of independentexperiments with 10 human and 4 rabbit blood donors. Statistically sig-nificant differences are indicated by asterisk (P < 0.05); all other comparisonsare nonsignificant.

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survival, which in turn is responsible for the pathology, orwhether PVL directly induces acute lung injury and lunginflammation. To address this issue, we conducted a time-courseexperiment in which rabbits were killed at 3, 6, and 9 h afterendotracheal instillation with wild-type strain, Δpvl strain, orsterile vehicle control. Infection with the wild-type and Δpvlstrains, but not instillation of sterile vehicle control, resulted insevere leukopenia by 9 h postinfection, evidenced by a decline inperipheral white-cell count to 8.6% and 16.3% of baseline val-ues, respectively (P = 0.311).Fig. 3A shows there was no difference in bacterial counts at 3,

6, or 9 h postinfection for wild-type or Δpvl-infected lungs,indicating that PVL-induced acute lung injury is independent ofbacterial survival/replication in the lung. In contrast, the LW/BWratio increased in a time-dependent manner with the wild-typestrain but not with the Δpvl strain (Fig. 3B). The gross appearanceof wild-type–infected lungs was markedly different from that of

Δpvl-infected lungs with extensive areas of necrosis and frothyedema brimming from the bronchi of wild-type–infected lung (Fig.3H). The protein content of bronchoalveolar lavage (BAL) fluid oflungs of rabbits infected with the wild-type strain, but not thoseinfected with Δpvl strain, increased over time (Fig. 3C), indicativeof an influx of plasma proteins into the alveolar space caused bydamage to the alveolar–endothelial barrier. Significantly greaterlevels of IL-8 andMCP-1 increased over time in lung and plasma ofwild-type–infected rabbits compared with Δpvl-infected rabbits(Fig. 3 D–G), and this finding correlated with significantly moreextensive PMN infiltration and destruction provoked by the wild-type strain as compared with the Δpvl strain (Fig. 3 I–K andTable S1).

PVL Causes Lung Inflammation and Injury by PMN-DependentMechanisms. We further hypothesized that lung injury is theresult of recruitment and subsequent lysis of PMNs, which damage

Fig. 2. PVL contributes to virulence of USA300 in a rabbit model of necrotizing pneumonia. (A) Kaplan–Meier survival curves for comparison of mortalityin rabbits infected via endotracheal instillation with increasing number of cfu of a SF8300 wild-type (wt) strain and its isogenic Δpvl mutant strain (n = 88total). Fifteen rabbits were randomized to receive one of the six inocula, which were blinded. Two rabbits had anesthesia-related deaths after ran-domization but before bacterial inoculation; these rabbits were excluded from subsequently analysis. Log-rank test was used to compute P values forcomparison of wild-type and Δpvl mutant strains at the same inocula. (B) Comparison of bacterial densities and (C) LW/BW ratio. (D) Kaplan–Meiersurvival curves for comparison of mortality in rabbits randomized for infection with blinded inocula containing wild-type, Δpvl mutant, or compΔpvlstrains (n = 45 total). (E ) Corresponding data on bacterial densities in the lung, (F) LW/BW ratio, (G) IL-8 per lung, and (H) MCP-1 per lung. In B, C, and E–H,filled symbols represent data from dead rabbits, and open symbols represent data from surviving rabbits that were killed 48 h postinfection. UnpairedStudent’s t test was used to compute two-sided P values for comparison of wild-type and Δpvl. (I) LukS-PV (in ng) per gram of lung tissue, as measured byELISA, after each extraction (open symbols, dashed lines) and final cumulative LukS-PV (in μg) per lung (filled symbols, solid lines); data represent mean ofthree necrotic lungs harvested from rabbits 12–39 h postinfection with either the wild-type strain (blue) or compΔpvl strain (green). LukS-PV was notdetected in lungs from rabbits infected with Δpvl mutant.

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the lung by releasing the contents of cytotoxic granules and/orreactive oxygenmetabolites. To test this hypothesis, we instilled 12μg each of LukS-PV andLukF-PV (i.e., 0.34 nmol of LukS-PV and0.32 nmol of LukF-PV), amounts comparable to that producedduring USA300 infection of rabbit lungs (Fig. 2I), endotracheallyinto lungs of normal rabbits and neutropenic (vinblastine-treated)rabbits (32). Because bothLukS-PVandLukF-PVare required forbiological activity (6, 7), each toxin subunit alone was used as anegative control. PurifiedLukS-PV+LukF-PV induced significantlevels of IL-8 and MCP-1 in the lungs compared with no cytokineinductionwhen either toxin subunit was administered alone (Fig. 4C andD). PurifiedLukS-PV+LukF-PV, but neither subunit alone,was sufficient to induce acute lung injury, which was accompaniedby a neutrophilic infiltrate, necrosis, diffuse alveolar hemorrhage,and pulmonary edema (i.e., an increase in the LW/BW ratio) innormal rabbits (Fig. 4 A–L). Purified LukS-PV+LukF-PVadministered to neutropenic rabbits did not cause an increase inIL-8 andMCP-1 levels (Fig. 4C andD), nor was there acute lunginjury (Fig. 4 H and L). Immunohistochemistry for IL-8 showedstaining of PMNs and to a lesser extent alveolar macrophages,but not epithelial or endothelial cells, in lungs of normal rabbits

instilled with LukS-PV+LukF-PV (Fig. 4K). Lungs of normalrabbits instilled with either toxin subunit alone (Fig. 4 I and J) didnot show staining for IL-8, whereas lungs of neutropenic rabbitsinstilledwith both toxin subunits showedweak staining for IL-8 inonly alveolar macrophages (Fig. 4L). This finding is consistentwith the reported specificity of PVL for myeloid cells and notother cell types (6) and also indicates that PMNs can amplify orperpetuate the acute inflammatory response by IL-8–mediatedrecruitment of additional PMNs into the inflamed lung. Takentogether, the data indicate that PMNs are critical for the devel-opment of PVL-induced lung inflammation and injury.

Concluding Comments. The striking epidemiological association ofPVL and severe necrotizing staphylococcal infections (1–5), in-cluding pneumonia, has prompted numerous studies to evaluatewhether PVL contributes to the disease process (10–18). Theinfection model for most of these studies was the mouse or the rat,which are relatively insensitive to the cytotoxic effects of PVL (6–8).To the extent that PVL target cell susceptibility is important inpathogenesis, experiments conducted in rodent models couldobscure the contribution of PVL and might fail to detect a key role

Fig. 3. Time-course of PVL-induced acute lung injury after endotracheal instillation with SF8300 wild-type (wt) strain, isogenic Δpvl mutant, or vehiclecontrol. Twenty-four rabbits each were randomized to receive either the wild-type or Δpvl mutant strain, which were blinded, and then eight rabbits chosenat random from each group were killed at 3, 6, or 9 h postinfection (n = 57 total). Three vehicle control-instilled rabbits were included for each time point. (A)Bacterial cfu per lung. (B) LW/BW ratio. (C) total protein concentration in BAL fluid. (D) IL-8 (in ng) per lung. (E) IL-8 (pg/mL) in plasma. (F) MCP-1 (in ng) perlung. (G) MCP-1 (ng/mL) in plasma. Unpaired Student’s t test was used to compute two-sided P values for between-group comparisons; statistical significantdifferences compared with wild type are indicated by an asterisk (P < 0.05); all other comparisons were nonsignificant. Photograph depicts lungs (H) andcorresponding H&E-stained sections (magnification 200×) harvested from rabbits at 9 h after endotracheal instillation of wild-type strain (I), isogenic Δpvlmutant (J), or vehicle control (K).

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Fig. 4. PVL-induced acute lung injury is mediated by PMNs. Normal rabbits were treated with endotracheal instillation of 12 μg of LukS-PV alone (n = 3),12 μg of LukF-PV alone (n = 3), or 12 μg each of LukS-PV and LukF-PV (n = 9); neutropenic rabbits were treated with 12 μg each of LukS-PV and LukF-PV (n = 6).All rabbits were killed 3 h after instillation to determine (A) LW/BW ratio, (B) total protein concentration in BAL fluid, (C) IL-8 (in ng) per lung, and (D) MCP-1(in ng) per lung. Unpaired Student’s t test was used to compute two-sided P values for between-group comparisons; differences that are statistically sig-nificant compared with wild type are indicated by an asterisk (P < 0.05); all other comparisons were nonsignificant. H&E-stained sections (E–H; magnification200×) or immunohistochemistry for IL-8 (I–L; magnification 400×) of lungs from normal rabbits instilled with LukS-PV only (E and I), LukF-PV only (F and J),LukS-PV and LukF-PV (G and K), or neutropenic rabbits instilled with LukS-PV and LukF-PV (H and L). Results in K showed deposition of brown reactionproduct (IL-8) in PMNs and to a lesser extent in alveolar macrophages.

Fig. 5. Mechanisms of PVL-induced acute lung injury and lung inflammation. Black arrows indicate observed events; gray arrows indicate postulated events.

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of PMNs, the major host cell target of PVL. Using a rabbit modelof necrotizing pneumonia, we found a clear role for PVL inpathogenesis and established a mechanistic basis for PVL-inducedacute lung injury and inflammation. Together with previouslypublished results, we postulate the following model of how PVLinduces acute lung inflammation and injury (Fig. 5). In this model,PVL-producing S. aureus gain access to the alveoli. At low bacte-rial burdens, the invading organisms eventually are cleared, sug-gesting that, if PVL has a role in establishing infection, otherpredisposing condition(s) such as antecedent or coincident influ-enza (2–5) are likely to be required also. At high bacterial burdens,PVL is produced in sufficient quantities to activate PMNs andmacrophages to release proinflammatory mediators, includingIL-8 (21, 28, 33, 34), thereby promoting recruitment of PMNs intothe inflamed lung. PVL then lyse PMNs, possibly resulting in therelease of granule contents, such as proteases and reactive oxygenmetabolites (20, 21, 33). In turn, the toxic products derived fromactivated or lysed PMNs could damage the alveolar epithelial andendothelial barriers, resulting in influx of fluid and protein fromthe vascular space into the airspace. Noncardiogenic pulmonaryedema and accompanying tissue injury and hemorrhagic lungnecrosis ensue, ultimately resulting in death. Knowledge ofmechanisms of lung injury gained from this model may lead to

new approaches to improve outcome in patients with severestaphylococcal pneumonia.

MethodsBacterial Strains. SF8300, a minimal-passaged USA300 clinical strain repre-sentative of the epidemic clone USA300-0114, and its isogenic Δpvl mutant,have been described previously (35). lukS-PV and lukF-PV were reintroducedinto their original chromosome sites in the Δpvl mutant by allelic replace-ment mutagenesis with plasmid pKOR1 (36) using the strategy outlined inFig. S1. PVL complementation was confirmed by PCR, DNA sequencing, andimmunoblotting (Fig. S1).

Other experimental details are provided in SI Text.

ACKNOWLEDGMENTS. We thank Paul M. Sullam andMichael Otto for criticaldiscussion and reading of the manuscript and Florence Couzon for technicalassistance. This work was supported by US Public Health Service NationalInstitute of Allergy and Infectious Diseases (NIAID) Grant AI070289 (to H.F.C.),by the Intramural Research Programof theNIAID,National Institutes ofHealth(to F.R.D.), and by University of California San Francisco Research Evaluationand Allocation Committee Pilot Award for Junior Faculty (to B.A.D.). P.T. wassupported by a grant from the Collège des Universitaires des Maladies Infec-tieuses et Tropicales and by the Pontchaillou University Hospital, Rennes,France. C.B., J.E., and G. L. were supported by Grant EC 222718 from the Euro-pean Community and by grants from Pfizer and LeoPharma. T.R.M. was sup-ported by Grant HL081764 from the National Heart, Lung and Blood Institute(NHLBI), G.Y.L. by Grant AI074832 from the NIAID, and M.A.M. by GrantsHL51854 from the NHLBI and 2P01A1053194 from the NIAID.

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