CD4� T cells and CXC chemokines modulatethe pathogenesis of Staphylococcus aureuswound infectionsRachel M. McLoughlin, Robert M. Solinga, Jeremy Rich, Kathleen J. Zaleski, Jordan L. Cocchiaro, Allison Risley,Arthur O. Tzianabos, and Jean C. Lee*

Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115

Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved May 11, 2006 (received for review October 13, 2005)

T cells are critical for the formation of intraabdominal abscesses byStaphylococcus aureus. We hypothesized that T cells modulate thedevelopment of experimental staphylococcal infections by control-ling polymorphonuclear leukocyte (PMN) trafficking. In models ofstaphylococcal s.c. abscess formation, hindpaw infection, and sur-gical wound infection, S. aureus multiplied in the tissues of WTC57BL�6J mice and elicited a marked inflammatory response. CD4�

�� T cells homed to the surgical wound infection site of WTanimals. In contrast, significantly fewer S. aureus were recoveredfrom the tissues of mice deficient in �� T cells, and the inflamma-tory response was considerably diminished compared with that ofWT animals. �� T cell receptor (���) mice had significantly lowerconcentrations of PMN-specific CXC chemokines in wound tissuethan did WT mice. The severity of the wound infection wasenhanced by administration of a CXC chemokine and abrogated byantibodies that blocked the CXC receptor. An acapsular mutantwas less virulent than the parental S. aureus strain in both the s.c.abscess and the surgical wound infection models in WT mice. Thesedata reveal an important and underappreciated role for CD4� ��T cells in S. aureus infections in controlling local CXC chemokineproduction, neutrophil recruitment to the site of infection, andsubsequent bacterial replication.

capsular polysaccharide � abscess

S taphylococcus aureus is the causative agent of a range ofserious human infections, including pneumonia, endocardi-

tis, and sepsis (1). In addition, this organism is a major cause oflocalized infections, such as s.c. abscesses, impetigo, and surgicalwound infections. The increasing prevalence of nosocomial andcommunity-acquired S. aureus infections has prompted studiesto better understand the host–pathogen interactions.

S. aureus produces a myriad of virulence factors that contributeto its ability to cause disease, allowing the organism to gain entryinto tissues, evade the host immune system, attach to host cells, andsecrete exoproteins and toxins. Cell wall components such aspeptidoglycan, teichoic acids, and capsule, as well as adhesins,proteases, exoproteins, and exotoxins, all promote the virulence ofthis pathogen (2–4).

In contrast, less is known about the host response to S. aureus andhow host factors influence the pathogenesis of staphylococcalinfections. Animal models of staphylococcal infections have beenused to investigate the interactions between S. aureus and the host.However, few recapitulate the major hallmarks of the disease as itoccurs in humans, because a large S. aureus inoculum [�106

colony-forming units (cfu)] is necessary to initiate infection, andlittle bacterial replication occurs in vivo. The amount of inoculumneeded to provoke disease can be reduced with the use of foreignbodies, such as Cytodex 1 beads (Sigma), to potentiate infection (5).

Polymorphonuclear leukocytes (PMNs) represent the host’s firstline of defense against invasion by S. aureus and are a criticaldeterminant in the outcome of staphylococcal infections (6). De-pletion of PMNs in vivo before bacterial challenge results in

overwhelming disease (7–9), and patients who are neutropenic orwho have congenital or acquired defects in PMN function are highlysusceptible to S. aureus infection (6). However, Gresham et al. (10)demonstrated that survival of S. aureus inside PMNs contributes tothe pathogenesis of staphylococcal infection in a murine peritonitismodel.

We have shown that T cells control the development of abscessesin an animal model of S. aureus intraabdominal abscess formationand that the S. aureus capsular polysaccharide (CP) activates CD4�

T cells in vitro (11). Because abscess formation depends on therecruitment of PMNs to the site of infection, we hypothesized thatT cells control the pathogenesis of S. aureus infection by modulatingthe PMN response in vivo. In this study, we investigated theinfluence of T cells on the pathogenesis of S. aureus infections inthree distinct animal models. Experiments in a clinically relevantsurgical wound infection model showed that T cells modulate thedevelopment of staphylococcal wound infections by controllinglocal CXC chemokine production, and ultimately PMN migrationto sites of infection. This work demonstrates that, although PMNsmay be beneficial to the host at the outset of S. aureus infection, theymay also play a deleterious role during the course of disease.

ResultsInfluence of T Cells in Subcutaneous Abscess Formation. Previousstudies (11, 12) have indicated that CD4� T cells play a role in thedevelopment of S. aureus intraabdominal abscesses. To determinewhether T cells might also influence other types of staphylococcalinfection, WT C57BL�6J and �� T cell receptor (TCR) (���)mice were challenged s.c. with 101 to 106 cfu of S. aureus PS80 [aserotype 8 CP (CP8) strain]. As shown in Fig. 1, WT mice exhibiteda clear dose response at low inocula (�3 � 103 cfu per mouse). Thefrequency of abscess formation reached 100% at inocula �3 � 103

cfu, and �106 cfu per abscess was recovered from these animals.Mice that were culture-negative developed smaller foreign-bodyresponses that contained Cytodex beads only. Gross pathologicinspection of the abscesses in WT mice revealed large vascularizedstructures with a thick fibrin wall (Fig. 1B). Histologic analysisrevealed that abscesses contained PMNs, bacteria, and Cytodexbeads (Fig. 1C). Confocal microscopic examination of abscessesfrom WT mice demonstrated that CD3� T cells had infiltrated thefibrin wall of these structures (data not shown). This observation isconsistent with previous studies demonstrating that T cells home toand comprise the wall of intraabdominal abscesses (13).

At inocula ranging from 3 � 102 to 3 � 104 cfu, �� TCR (���)mice had fewer culture-positive abscesses and significantly fewer cfu

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: cfu, colony-forming units; PMN, polymorphonuclear leukocyte; CP, capsularpolysaccharide; TCR, T cell receptor; KC, keratinocyte-derived cytokine; MIP, macrophageinflammatory protein; CXCR, CXC receptor.

*To whom correspondence should be addressed. E-mail: jean.lee@channing.harvard.edu.

© 2006 by The National Academy of Sciences of the USA

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per abscess than WT animals (Fig. 1A). Most of the �� TCR (���)mice challenged with �104 cfu were culture-negative. �� TCR(���) mice that were culture-positive developed a tissue responsethat differed from that of a classic abscess. These structures weresmaller and lacked a thick fibrin wall (Fig. 1B). Histologic analysisrevealed that the structures were composed of hyperproliferativefibroblasts characteristic of granulation tissue found in a foreign-body response but lacked significant PMN infiltration (Fig. 1D).

Influence of T Cells in the Hindpaw Infection Model. Rich and Lee (14)described an S. aureus infection model in the hindpaw of nondia-betic and diabetic mice. We inoculated a hindpaw of WT and ��TCR (���) mice with 106 cfu of PS80 and euthanized the miceafter 5 days. Quantitative culture results revealed that WT mice had

significantly (P � 0.0178) more S. aureus recovered from thehindpaws than did �� TCR (���) animals: 5.24 � 0.43 vs. 2.19 �0.44 cfu�g of tissue, respectively (mean log10 cfu � SEM).

Development of a Surgical Wound Infection Model. Our initial ex-periments were aimed at optimizing the inoculum needed toprovoke a staphylococcal surgical wound infection in mice. Inoc-ulation of surgical wounds with �103 cfu of S. aureus PS80 resultedin a localized purulent infection on day 3 that showed markedinflammation on gross pathological inspection. Challenge withinocula �106 cfu resulted in a severe and purulent infection. Tissuesfrom mice inoculated with 101 to 108 cfu of PS80 were culturedquantitatively on days 3 or 6. As few as 10 cfu of S. aureus initiatedinfection, and the bacterial burden increased �1,000-fold over a3-day period in mice challenged with 102 or 103 cfu of S. aureusstrains PS80, Reynolds (a CP5 strain), or COL (a methicillin-resistant CP5 strain) (Fig. 2A). The tissue bacterial load was notsignificantly different between 3 and 6 days after inoculation.Animals infected with 2 � 106 cfu of S. aureus PS80 in the absenceof a sutured wound yielded �1�100th as many S. aureus as animalswith sutured wounds (data not shown). A time-course experimentin mice challenged with 103 cfu of S. aureus PS80 revealed that thebacterial burden in the wounded tissue achieved maximum levels(106 to 107 cfu�g of tissue) between 3 and 12 days after inoculation(Fig. 2B). The infection declined by day 18, when two of four micehad cleared the infection.

Influence of T Cells on Surgical Wound Infections. To determinewhether T cells influenced the pathogenesis of S. aureus-inducedsurgical wound infections, initial experiments were performed withan inoculum of 40 cfu of strain PS80. Similar to our findings withthe abscess and hindpaw infection models, WT mice had signifi-cantly (P � 0.0445) more S. aureus recovered from the infectedwound tissue than did �� TCR (���) animals on day 3: 5.05 � 0.60vs. 2.59 � 0.76 cfu�g of tissue, respectively (mean log cfu � SEM).Mice deficient in different T cell subsets were then used todetermine the phenotype of T cells that contribute to the patho-genesis of S. aureus wound infections. CD8-deficient mice chal-lenged with 102 cfu had 6.88 � 0.67 log cfu�g of tissue recoveredfrom wounds at day 3, similar to the bacterial burden recoveredfrom WT mice (6.81 � 0.23). In contrast, mice deficient in �� TCRor CD4 or having T cells lacking the TCR� chain had significantlyfewer bacteria at the wound site 3 days after inoculation with 102 cfuof S. aureus than did WT mice (Table 1). Likewise, the T cell-deficient mice had fewer S. aureus recovered from the wounds thandid WT animals on day 6, and this difference was significant (P �0.035) for the CD4 (���) mice. WT and T cell-deficient animalschallenged with �103 cfu of S. aureus had similar numbers ofbacteria recovered from the wounds on both days.

Fig. 1. Role of T cells in s.c. abscesses induced by S. aureus PS80. (A)Quantitative cultures of abscesses from WT or �� TCR (���) mice weresignificantly different at inocula of 3 � 104 (P � 0.026) and 3 � 103 (P � 0.038)cfu per mouse. The number of animals that were culture-positive in eachgroup is indicated. (B) WT mice challenged with S. aureus formed definedabscesses with a thick fibrin wall (fw). In contrast, �� TCR (���) mice devel-oped small foreign-body responses (fb). (C) Histological analysis showed fi-brin-encased (fw) abscesses in WT mice and significant leukocyte infiltration(inf). Cytodex beads (cb) are visible in the abscess tissue. (D) The foreign-bodyresponse seen in the �� TCR (���) mice consisted primarily of granulationtissue (gt).

Fig. 2. S. aureus wound infection model dose–response curve (A) and time course (B). (A) WT mice were challenged with increasing inocula of strain PS80(circles), Reynolds (squares), or COL (triangles) and euthanized at either 3 days (open symbols) or 6 days (filled symbols). (B) WT mice were challenged with 103

cfu of S. aureus PS80 and euthanized at the designated time points.

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Histopathology of Surgical Wound Tissues. Examination of thewound tissues from WT mice challenged with PBS revealed aspectsof the normal host response to a foreign body. PMNs were detectedat the suture site by 24 h after surgery. By day 3 the PMN infiltrationwas accompanied by a densely staining infiltrate of hyperprolifera-tive fibroblasts associated with granulation tissue (Fig. 3A). Thisobservation is consistent with the normal process of wound repairthat follows tissue trauma. More granulation tissue and fewerPMNs were observed at the wound site by day 6 (data not shown),indicating that the normal wound healing process was under way.

Challenge of WT animals with 102 cfu of S. aureus resulted innumerous PMNs infiltrating the wound site 24 h after surgery.Additional PMNs entered the wound site by day 2 and were foundaround the suture and in the surrounding muscle tissue. Mononu-clear cells were observed infiltrating the wound site on day 2. By day3, wounds exhibited an intense, purulent infection with numerousPMNs and mononuclear cells visible near the suture and in thesurrounding tissue (Fig. 3B). The cellular infiltrate in infectedtissues was greater than that observed in PBS-challenged mice ateach time point. S. aureus-infected wounds on day 6 were histo-logically similar to those from day 3, an indication that the hostresponse had not abated. Mice challenged with 103 cfu of S. aureusPS80 exhibited a more severe infection than mice given 102 cfu, butthe time course of histopathologic changes was similar (data notshown).

Histologic examination of surgical wound tissues from �� TCR(���) mice on day 3 revealed less inflammation in response tochallenge with 102 (Fig. 3C) or 103 cfu of S. aureus compared withWT animals. Tissues from �� TCR (���) mice showed reducedPMN infiltration and fewer mononuclear cells around the suturesite and in the surrounding muscle tissue compared with that seenin WT animals. Granulation tissue associated with normal woundrepair was noted in tissues from �� TCR (���) mice.

T Cells Traffic to S. aureus-Infected Wounds. Confocal microscopywas used to investigate the types of cells that infiltrated the woundsite during S. aureus infection. WT mice were challenged with PBSor 102 cfu of S. aureus PS80, and the wounds were harvested dailyuntil day 6 after surgery. Mice challenged with PBS showed somePMN infiltration at the wound site, but no �� T cells were observedon day 3 (Fig. 3D). Infiltration of PMNs occurred within the first24 h of infection in WT mice challenged with S. aureus, whereas Tcells could be detected by day 2 after inoculation. Cellular infiltra-tion by T cells and PMNs achieved maximal levels on day 3 (Fig.3E). PMNs, T cells, and bacteria were present around the suturesand in the surrounding muscle tissue. The �� T cells in the woundtissues (Fig. 3E) stained positive for CD4 but not for CD8 (data notshown).

Confocal microscopic analysis of tissues from �� TCR (���)mice challenged with 102 or 103 cfu of S. aureus PS80 confirmed our

Table 1. Bacterial burden in wounds of T cell-deficient mice challenged with 102 cfuof S. aureus

Day

Log10 cfu of S. aureus per g of tissue

WT �� TCR (�/�) TCR� (�/�) CD4 (�/�)

3 6.81 � 0.23(n � 7)

4.89 � 0.66(n � 7; P � 0.028)

2.85 � 0.95(n � 4; P � 0.027)

5.34 � 0.57(n � 11; P � 0.0327)

6 5.90 � 0.87(n � 7)

4.21 � 0.70(n � 8; P � 0.158)

2.92 � 1.06(n � 4; P � 0.074)

3.33 � 0.65(n � 11; P � 0.035)

Results are presented as mean � SEM. P values are vs. WT.

Fig. 3. Histopathology and confocal microscopy of wound tissues. (Magnification: �100.) Wounds were inoculated with PBS or 102 cfu of S. aureus PS80 andharvested on day 3. Tissue sections were processed for histology (A–C) or stained with mAbs specific for PMNs (red) and �� T cells (green) and polyclonal Absto S. aureus PS80 (blue) for examination by confocal microscopy (D–F). PBS-challenged wounds (A and D) showed the influx of some PMNs and monocytes, butno T cells. Wounds inoculated with S. aureus (B and E) showed a marked inflammatory response characterized by infiltration of PMNs and mononuclear cells.(Inset) T cells were observed infiltrating to the infected suture site. Surgical wounds in �� TCR (���) mice challenged with S. aureus (C and F) revealed a lessintense cellular infiltrate with relatively few PMNs and mononuclear cells present.

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histologic findings that T cell deficiency alters the cellular hostresponse. Compared with WT mice, �� TCR (���) mice showeda mitigated inflammatory response in the wounds on day 3, withnotably fewer PMNs and mononuclear cells present (Fig. 3F).

CXC Chemokine Response in Staphylococcal Wound Infections. PMNsmigrate to sites of infection in response to chemoattractants such asthe CXC chemokines. Levels of keratinocyte-derived cytokine(KC�CXCL1) and macrophage inflammatory protein 2 (MIP-2�CXCL8) were assessed in wounds inoculated with 102 cfu of S.aureus PS80 or PBS at 6, 24, and 48 h after surgery. Challenge ofWT mice with S. aureus resulted in significantly (P � 0.036) higherKC levels 6 h after challenge compared with mice given PBS (Fig.4A). KC tissue levels diminished with time, reaching baseline levelsby 24 h after inoculation. In contrast, the levels of MIP-2 reacheda maximum at 48 h in the S. aureus-infected tissues (Fig. 4B), atwhich point the MIP-2 level was significantly (P � 0.026) greaterthan that found in control animals. No further increase in MIP-2levels was observed in samples analyzed at 72, 96, or 120 h (data notshown). To determine the influence of T cells on CXC chemokineproduction in response to S. aureus wound infection, WT and ��TCR (���) mice were challenged with 102 cfu of S. aureus PS80.KC concentrations in wound tissue from �� TCR (���) mice weresignificantly (P � 0.004) lower at 6 h compared with tissue levels inthe WT group (Fig. 4C). Likewise, MIP-2 concentrations weresignificantly (P � 0.0007) decreased in the �� TCR (���) mice at48 h compared with WT mice (Fig. 4D).

Role of CXC Chemokines in S. aureus Wound Infections. KC and MIP-2bind to a common CXC receptor (CXCR2) on PMNs, and block-ade of this receptor in vivo results in decreased PMN recruitmentto sites of inflammation (15). Administration of CXCR2 antibodiesto WT mice resulted in a significantly (P � 0.001) reduced bacterialburden in S. aureus-infected wounds compared with administrationof an IgG control antibody (Fig. 4E).

The specific role of MIP-2 was addressed by challenging �� TCR(���) mice with S. aureus PS80 and then treating them with a CXCchemokine (MIP-2) or a CC chemokine MIP-1 (MIP-1��CCL3) 4and 24 h after surgery. Administration of MIP-2 produced asignificantly (P � 0.004) greater bacterial burden at the suture sitethan administration of the CC chemokine MIP-1� (Fig. 4F).

Role of the Capsule in the Pathogenesis of S. aureus Infections. Thecapsule of S. aureus has been implicated as a virulence factor for thisorganism (16–19). CP8 activates CD4� T cells in vitro, and activatedT cells induce intraabdominal abscesses when transferred to naı̈veanimals (11). Therefore, we assessed whether the type 8 capsuleinfluenced the pathogenesis of s.c. abscess formation or experi-mental staphylococcal wound infection. The acapsular mutantRMS-1 was less virulent than the parental strain PS80 in bothinfection models (Table 2). WT mice inoculated with 102 or 103 cfuof RMS-1 had significantly (P � 0.05) fewer S. aureus recoveredfrom the infection sites than mice challenged with strain PS80. Incontrast, strain PS80 and mutant RMS-1 showed similar virulencein �� TCR (���) mice inoculated with 102 cfu of S. aureus (meanlog cfu�g of tissue � SEM: 5.32 � 0.31 and 4.84 � 0.52, respec-tively). MIP-2 and KC concentrations in WT mice infected with the

Fig. 4. Role of chemokines in S. aureus wound infections. (A) WT micechallenged with 102 cfu of S. aureus PS80 showed increases in KC levels at 6 and24 h compared with mice given PBS. (B) MIP-2 levels were significantly greaterat the 48-h time interval in the S. aureus group than in the PBS group. (C) Afterchallenge with 102 cfu of S. aureus PS80, peak tissue concentrations of KC weresignificantly lower in �� TCR (���) mice than in WT mice at 6 h. (D) MIP-2 levelswere significantly decreased in the �� TCR (���) mice at 48 h. (E) Quantitativecultures in WT mice were performed on day 3 after challenge with 102 cfu ofS. aureus and i.p. administration of antibodies 1 h before and 6 h after surgery.(F) �� TCR (���) mice were challenged with 43 cfu of S. aureus PS80, and MIP-2or MIP-1� (400 ng) was administered directly into the wound 4 and 24 h aftersurgery. Cultures were quantified on day 3.

Table 2. The S. aureus capsule promotes virulence in mouse models of S. aureus s.c. abscess formation andsurgical wound infection

Inoculum,log10 cfu/mouse

Subcutaneous abscess Wound infection

Log10 cfu of S. aureus per abscess

P

Log10 cfu of S. aureus per g of tissue

PPS80 RMS-1 (acapsular) PS80 RMS-1 (acapsular)

3 6.29 � 0.4(n � 6)

4.92 � 0.41(n � 6)

0.049 7.01 � 0.19(n � 17)

6.06 � 0.34(n � 20)

0.021

2 3.7 � 0.63(n � 12)

2.13 � 0.51(n � 14)

0.070 6.36 � 0.45(n � 14)

4.84 � 0.52(n � 28)

0.013

Results are presented as mean � SEM.

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acapsular mutant RMS-1 were not significantly different from thosein PS80-infected mice (data not shown).

DiscussionAn important aspect of the host innate immune response to S.aureus is opsonophagocytic killing by PMNs. The essential role ofphagocytic killing in host clearance of S. aureus is apparent becausepatients who are neutropenic or have defects in PMN functionsuffer from recurrent staphylococcal infections. In contrast, littleinformation is available on how various T cell populations influencethe outcome of staphylococcal infections. We reported that the S.aureus CP activates CD4� T cells in vitro and that activated T cellsinduce intraabdominal abscesses when transferred to naı̈ve animals(11). These results led us to consider the T cell as an importantmediator of the host response to this organism.

We evaluated the role of T cells in modulation of host innateimmunity in three different animal models of S. aureus infection.Whereas WT mice formed well-defined abscesses at inocula as lowas 103 cfu, �� TCR (���) mice were impaired in their ability todevelop defined abscesses. Few PMNs were noted in tissue sectionsfrom �� TCR (���) mice, and significantly fewer bacteria wererecovered from the infection site of the mutant animals comparedwith WT animals. Similarly, in the hindpaw infection model,challenge of �� TCR (���) mice resulted in a significantly lowerbacterial burden in the infected tissue than in WT animals. Thesedata are concordant with the results of our earlier studies indicatingthat T cells control the development of S. aureus intraabdominalabscesses (11, 12).

These experimental findings were investigated in greater depth ina wound infection model that mimics S. aureus infection in a surgicalsetting. As few as 10 cfu initiated infection in this model, andbacterial numbers increased in vivo by 3 to 4 orders of magnitude.The infection remained localized; the mice did not become bacte-remic even when �106 cfu were delivered to the wound.

Few studies have critically assessed the contribution of thehost response to the pathogenesis of staphylococcal woundinfections. Confocal microscopic analyses of S. aureus-infectedwounds from WT mice confirmed histologic findings that PMNsinfiltrated the wound tissue by 24 h after bacterial inoculation.�� CD4� T cells were observed at the wound site on day 2 andwere abundant in the tissues by day 3. Compared with WT orCD8-deficient mice, fewer bacteria were cultured from thewound site of �� TCR-, CD4-, and TCR� chain-deficient miceat 3 and 6 days after challenge with 102 cfu of S. aureus. Amarkedly reduced PMN infiltrate was observed at the suture siteof �� TCR-deficient mice compared with WT mice, suggestingthat CD4� �� T cells contribute to the pathogenesis of staph-ylococcal wound infections by controlling PMN infiltration.

Because CXC chemokine levels were significantly lower in ��TCR (���) mice than in WT animals, it is likely that T cells controlPMN recruitment to the infected site through modulation of CXCchemokine levels and subsequent PMN migration to surgicalwounds. Antibody blockade of the CXCR2, which binds bothMIP-2 and KC in vivo, significantly reduced the bacterial burden atthe infection site. Conversely, administration of MIP-2 directly intothe wound at the time of challenge resulted in significantly greaterbacterial counts in infected wounds than in wounds treated with theCC chemokine MIP-1�. These data clearly demonstrate that CXCchemokines modulate the pathogenesis of staphylococcal woundinfections.

T cells have been shown to regulate neutrophil trafficking in theclinical setting as well. In patients suffering from acute generalizedexanthematous pustulosis, a severe drug hypersensitivity resultingin the formation of neutrophil-laden pustules in the skin, CD4� Tcells produce IL-8 and regulate neutrophilic infiltration to theinflammatory site (20). Phenotypic analysis of these CXC-producing T cells revealed that they express a chemokine receptorand cytokine profile associated with a T helper 1 effector memory

phenotype. Further analysis is needed to identify the specificphenotypes of the T cells involved in regulating neutrophil traf-ficking during the course of an S. aureus wound infection.

Gresham et al. (10) demonstrated that local release of CXCchemokines promoted PMN migration but also resulted in asituation in which PMNs internalized but did not kill S. aureus. Theauthors postulate that CXC chemokines increase the persistence oflive S. aureus within PMNs and provide the intracellular bacteria aniche where they are protected from competent phagocytes. Wehave shown in preliminary experiments that administration ofMIP-2 into S. aureus-infected wounds of mice results in a greaternumber of viable bacteria internalized within PMNs than is the casein MIP-1�-treated animals (unpublished data). The potentiallydeleterious role of PMNs in staphylococcal disease is likely afunction of the dual biologic activities displayed by CXC chemo-kines: they recruit PMNs to the infection site, but they may alsodelay PMN apoptosis and clearance by macrophages (21, 22). Theseevents result in prolonged PMN infiltration and failure to clearreplicating organisms, thus promoting chronic infection. This sce-nario is consistent with that of patients with periodontal disease,inasmuch as PMNs harvested from their oral abscesses or gingivitissites show a significantly reduced ability to kill Porphyromonasgingivalis (23).

Both PMNs and T cells produce CXC chemokines, and localMIP-2 and KC levels are increased in animal models of S. aureus-induced brain abscesses and lung empyema (24–26). CD4� T cellsplay a critical role in the pathogenesis of experimental S. aureusempyema (26), inasmuch as CD4 (���) mice had significantlyfewer PMNs and lower concentrations of KC and MIP-2 in thepleural space than WT animals. Mice infected with S. aureus in oursurgical wound model responded with early (6 h) KC production,whereas maximal MIP-2 production occurred at 48 h. These dataare consistent with a previous study that showed a distinct temporalpattern of expression for these two functionally similar chemokinesin a surgical injury model (27). The authors postulate that otherinflammatory mediators at the wound site may differentially reg-ulate the expression of KC and MIP-2. KC may be predominantlyinvolved in directing initial PMN recruitment to the infection site,whereas MIP-2 may function in later aspects of the immuneresponse to injury, e.g., CXC chemokines have been implicated inwound healing (28).

We showed previously (11) that S. aureus CP8 activates CD4� Tcells in vitro. Reports of other biological properties of CP8 are scant.The CP5 produced by S. aureus promotes virulence in numerousmodels of staphylococcal infection, including bacteremia, abscessformation, and septic arthritis (11, 17, 18, 29, 30), and protects thebacterium from opsonophagocytosis in vitro (17, 18, 29). Wegenetically modified the serotype 5 strain Reynolds to express CP8.Reynolds (CP8) was more virulent in a mouse bacteremia modeland in phagocytic killing assays than an acapsular mutant but lessvirulent than Reynolds (CP5) (18). In the present study, the impactof S. aureus CP8 production was assessed in the s.c. abscess and thesurgical wound infection models. The acapsular mutant RMS-1 wassignificantly less virulent than strain PS80 in each model. Incontrast, the two strains showed equivalent virulence in �� TCR(���) mice, which are unresponsive to S. aureus CP. Clearly,bacterial factors other than CP8 influence the outcome of staph-ylococcal infections because even the acapsular mutant causedinfection at inocula �103 cfu (data not shown). S. aureus wallteichoic acid (produced by both strains) was as potent as CP8 ininducing intraabdominal abscesses in rats (11). Chemokine levelswere equivalent in WT mice challenged with either RMS-1 or PS80.Other Gram-positive cell wall components common to both strains,such as peptidoglycan and lipoteichoic acid, have been shown toelicit a host inflammatory response in WT mice (31).

In summary, T cells modulate the pathogenesis of S. aureusinfection in three different animal models. A low-inoculum, clini-cally relevant model of surgical wound infection was developed to

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study the role of T cells in disease progression and to characterizethe host response to infection. The data demonstrate that T cellsorchestrate the outcome of this infection through regulation ofPMN infiltration and CXC chemokine production at the site. Ourstudy underscores the importance of T cells in the control of certainstaphylococcal infections, possibly through modulation of PMNfunction during the host response.

Materials and MethodsBacterial Strains. S. aureus strains PS80, Reynolds, and COL aredescribed in ref. 11. Mutant RMS-1 is an isogenic acapsular mutantof strain PS80 that was derived by allelic replacement mutagenesiswith the temperature-sensitive vector pAP1.2 (18). Staphylococciwere cultivated for 24 h at 37°C on Columbia agar (Difco) with 2%NaCl.

Mouse Models of S. aureus Infection. Male WT C57BL�6J andcongenic gene-deficient mice were obtained at 6–8 weeks of agefrom The Jackson Laboratory. Animal experiments were per-formed in accordance with the guidelines of the Harvard MedicalSchool Standing Committee on Animals.Subcutaneous abscess model. This model is described in ref. 30. Micewere anesthetized with 100 mg�kg ketamine and 10 mg�kg xylazine,and the hair on their flanks was removed. The mice were injecteds.c. in each flank with 0.2 ml of an S. aureus�Cytodex bead (Sigma)mixture containing 107 to 101 cfu. On day 4, the mice wereeuthanized and the abscesses were excised, homogenized, andcultured quantitatively.Hindpaw infection model. This model of S. aureus infection is de-scribed in ref. 14. A 10-�l suspension of S. aureus containing 106 cfuwas injected into the plantar proximal aspect of the hindpaw. Themice were euthanized on day 5, and their hindpaw tissue washomogenized and cultured quantitatively.Wound infection model. In this model, mice were anesthetized andtheir right thighs were shaved and disinfected. The thigh muscle wasexposed, and a 1-cm incision was made in the muscle to the depthof the bone. The incision was closed with one 4-0 silk suture, and5 �l of an S. aureus suspension or PBS was introduced into themuscle incision under the suture. The skin was closed with fourProlene sutures (Ethicon, Somerville, NJ). At designated intervalsafter surgery, the mice were euthanized and the muscle tissue was

excised, weighed, and homogenized, and S. aureus were culturedquantitatively.

Histologic and Confocal Analysis of Tissues. Wound tissue was ex-cised, fixed in formalin, embedded in paraffin, and stained withhematoxylin and eosin for microscopic examination. For confocalmicroscopy, tissue sections were dewaxed, permeabilized with0.05% saponin, and incubated with normal goat serum to inhibitnonspecific binding. Tissue sections were stained with murine-specific antibodies (Ly-6G clone 1A8 for PMNs, clone H57-597 for�� TCR, and GK1.5 for CD4; BD Pharmingen) or with MCD0820for CD8� (CalTag, South San Francisco, CA). Slides were viewedwith a multiphoton microscope with a krypton�argon laser (Bio-Rad) for standard confocal microscopy and a mode-locked titani-um-sapphire laser (Spectra-Physics) for multiphoton imaging in-terfaced with a Zeiss Axiovert microscope.

Chemokine Analysis. Wound tissues were excised and homoge-nized in 500 mM NaCl�50 mM Hepes, pH 7.4, containing 0.1%Triton X-100, 0.5 �g�ml leupeptin, 1 mM PMSF, and 0.02%NaN3. The homogenates were subjected to a freeze�thaw cycleand centrifuged at 6,000 � g for 20 min at 4°C. Concentrationsof KC and MIP-2 in the cleared supernatants were determinedby ELISA (R & D Systems).

Antibody and Chemokine Administration. A goat polyclonal antibodyto murine CXCR2 is described in refs. 15 and 32. The CXCR2antibody (100 �g) or a control IgG antibody was administered i.p.to WT mice 1 h before and 6 h after surgery. Wounds wereinoculated with 102 cfu of S. aureus PS80 and harvested on day 3 forquantitative culture. For chemokine administration, 400 ng ofMIP-2 or MIP-1� (R & D Systems) was injected directly into thewound site of �� TCR (���) mice 4 h and 24 h after inoculationwith 30 cfu of S. aureus PS80. Tissues were harvested on day 3 forquantitative culture.

Statistical Analyses. Quantitative culture results and tissue chemo-kine levels were compared by the Welch modification of theunpaired Student t test.

We thank Dr. Vincent Carey for advice regarding statistical analyses.This work was supported by National Institutes of Health GrantsAI52397 (to A.O.T.) and AI29040 (to J.C.L.).

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