Increased Resistance to Staphylococcus aureus Endophthalmitis inBALB/c Mice: Fas Ligand Is Required for Resolution of Inflammationbut Not for Bacterial Clearance

Norito Sugi, Emily A. Whiston, Bruce R. Ksander, Meredith S. Gregory

The Schepens Eye Research Institute, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA

FasL was recently shown be required for bacterial clearance in C57BL/6 mice that express the FasL.1 allotype. The FasL.2 allotypeis expressed in BALB/c mice and exhibits increased binding affinity to and increased cytotoxic activity against Fas� target cells.Therefore, we hypothesized that BALB/c mice would be more resistant to Staphylococcus aureus-induced endophthalmitis. Totest this hypothesis, C57BL/6, BALB/c, and BALB(gld) mice received intravitreal injections of 2,500 CFU of S. aureus (RN6390).Clinical examinations, electroretinography (ERG), histology, and bacterial quantification were performed at 24, 48, 72, and 96 hpostinjection. The myeloperoxidase (MPO) assay was used to quantitate neutrophil infiltration. At 96 h postinfection, 86% ofC57BL/6 mice presented with complete destruction of the eye, compared to only 29% of BALB/c mice with complete destruction.To our surprise, in the absence of Fas ligand, BALB(gld) mice showed no difference in bacterial clearance compared to BALB/cmice. However, histology and ERG analysis revealed increased retinal damage and significant loss of retinal function. MPO anal-ysis revealed equal numbers of neutrophils in BALB(gld) and BALB/c mice at 24 h postinfection. However, at 48 h, the neutro-phil numbers remained significantly elevated in BALB(gld) mice, correlating with the increased retinal damage observed inBALB(gld) mice. We conclude that the increased resistance to S. aureus induced endophthalmitis in BALB/c mice is not depen-dent upon the FasL. However, in contrast to C57BL/6 mice, FasL is required for resolution of inflammation and protecting hosttissue from nonspecific damage in BALB/c mice.

Bacterial endophthalmitis is a rare but serious infection of theposterior segment of the eye that can occur as a complication

following ocular surgery or globe-penetrating injury, often result-ing in significant retinal damage and loss of vision (1–4). Theretinal damage occurs directly, as a consequence of bacterial tox-ins and cytolytic factors that lyse cells and damage host tissue, aswell as indirectly, as a consequence of host inflammation thatresults in bystander damage of normal tissue (5–7). The extent ofdamage is dependent upon variables that are interconnected, thevirulence of the infecting organism, and the severity of the hostinflammatory response. Consequently, in spite of aggressive ther-apy with broad-spectrum antibiotics aimed at the infecting patho-gen, endophthalmitis often leads to partial or complete loss ofvision due to the damage of photoreceptor cells and/or retinaldetachment (1, 2). Thus, more successful treatment of bacterialendophthalmitis will require not only the elimination of the in-fecting bacteria but also proper regulation of the host immuneresponse to minimize nonspecific bystander destruction of nor-mal tissue.

The host immune response to intraocular infection is multi-factorial, with the clearance of a bacterial infection dependentupon the early recruitment and activation of neutrophils (8). Sev-eral studies have demonstrated the importance of cytokines andchemokines in a timely recruitment and activation of neutrophils(9). More recently, however, Fas ligand has also been shown to becritical in the clearance of bacterial endophthalmitis and may playa direct role in neutrophil recruitment and activation of neutro-phils (10), supporting earlier reports that identified FasL as a po-tent chemoattractant for neutrophils (11, 12). Fas ligand is con-stitutively expressed in tissues of the normal eye and has beenshown to play a central role in the maintenance of the immune-privileged environment by inducing apoptosis in infiltrating in-

flammatory cells (13). On the other hand, in a model of Staphylo-coccus aureus-induced endophthalmitis, it has been shown thatwhile normal mice readily cleared an infection of S. aureus (500CFU), mice deficient in Fas ligand were unable to clear the same-size inoculum (10). In the absence of FasL, bacteria grew morerapidly and fewer neutrophils were recruited to the site of infec-tion, suggesting that FasL is important for early activation of in-nate immunity within the eye (10).

Findings from our laboratory support the concept that FasL isimportant in triggering the early phase of innate immunity. Wedemonstrated that FasL engagement of Fas� macrophages notonly induces apoptosis but also induces the production of neutro-phil chemotactic factors (14). Moreover, using an ocular tumormodel, we demonstrated that overexpression of the membraneform of FasL within the immune-privileged eye induced a potentneutrophil-mediated inflammatory response, leading to the ter-mination of ocular immune privilege (15, 16). Together, thesestudies suggest that a more efficient and/or rapid engagement of

Received 22 April 2013 Returned for modification 15 May 2013Accepted 29 March 2013

Published ahead of print 8 April 2013

Editor: B. A. McCormick

Address correspondence to Meredith S. Gregory,meredith.gregory@schepens.harvard.edu.

N.S. and E.A.W. contributed equally and should be considered co-first authors.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00405-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00405-12

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the Fas receptor on cells resident within the eye will trigger aquicker innate-mediated inflammatory response, acceleratepathogen clearance, and ultimately increase resistance to bacterialendophthalmitis.

In order to test whether increased activation of the Fas receptorby FasL leads to more efficient bacterial clearance, we examinedwhether a polymorphism of murine Fas ligand, which increasesthe specific activity of FasL, affects resistance to S. aureus-inducedendophthalmitis. Sequence analysis of FasL cDNA from severalstrains of mice determined that inbred mice segregate into twoFasL allotypes: mFasL.1 and mFasl.2 (17). The mFasL.2 allotypeexhibits 9-fold-higher binding affinity to the Fas receptor and cy-totoxic activity against Fas� target cells than mFasL.1. Polymor-phisms in the FasL gene have also been found in humans and havebeen associated with increased risk of some cancers (18–20).However, it is not known how these FasL polymorphisms mayinfluence the immune response and susceptibility to infection inhumans. Moreover, the C57BL/6 mice used in previous reportsexpress the mFasL.1 allotype (weaker binding affinity), whereasBALB/c mice express the mFasL.2 allotype (stronger binding af-finity). Motivated by our data showing that FasL engagement ofthe Fas receptor induces not only apoptosis but the production ofproinflammatory mediators and neutrophil chemotactic factors,we asked whether BALB/c mice that express the mFasL.2 allotypedevelop an innate-mediated inflammatory response that is morerobust than mice expressing the mFasL.1 variant and if this wouldresult in increased resistance to bacterial endophthalmitis. Thedata presented herein indicate that BALB/c mice are indeed moreefficient at bacterial clearance, resulting in increased resistance toendophthalmitis compared to that of C57BL/6 mice. However,the increased resistance in BALB/c mice was not dependent uponthe Fas-FasL pathway. Our results indicate that in contrast toC57BL/6 mice, the Fas-FasL pathway is not required for bacterialclearance in BALB/c mice. Moreover, neutrophil infiltration is notdependent upon FasL expression, as shown previously in C57BL/6mice. Rather in BALB/c mice, the Fas-FasL pathway is critical inresolution of inflammation and protecting the retina from non-specific bystander tissue damage.

MATERIALS AND METHODSAnimals. Female and male C57BL/6J mice, BALB/cJ mice, and BALB(gld)(Cpt.C3-Tnfsf6gld) mice were purchased from The Jackson Laboratory(Bar Harbor, ME). All animals were treated according to the Associationfor Research in Vision and Ophthalmology Resolution on the Use of An-imals in Research. All procedures involving mice were approved by theSchepens IACUC.

Induction of endophthalmitis. Mice received an intravitreal injectionof 0.5 �l of sterile physiological saline (Hospira, Lake Forest, IL), in which500, 2,500, 5,000, or 10,000 CFU of S. aureus (strain RN6390) was sus-pended, into the left eye, according to a previously described protocol(10). Briefly, an intravitreal injection of S. aureus, just posterior to thelimbus-parallel conjunctival vessels, was performed using a borosilicatemicrocapillary pipette pulled to a tip size of 50 �m. Dilation of the pupilwith 1% tropicamide (Alcon Laboratories, Inc., Fort Worth, TX) alloweddirect visualization of the needle during the injection. During injection, ifthe lens or retina was damaged, the mouse was removed from the study. Inaddition, any mice that developed hemorrhage at the time of injectionwere also removed from the study. The right eye of each mouse was un-treated and served as an internal control for electroretinography studies.Before injection, S. aureus cells were grown in standard Bacto brain heartinfusion broth (BD Bioscience, Sparks, MD) and diluted in phosphate-

buffered saline (PBS) during log phase to the appropriate concentration(500, 2,500, 5,000, or 10,000 CFU/0.5 �l).

Clinical scoring. At 24, 48, 72, and 96 h following injection, clinicalexams were performed by slit lamp microscopy. Mice were given a clinicalscore of 0 to 3, as previously described (21). A score of 0 was equivalent toa clear anterior chamber, clear vitreous, and clear view of the retina; ascore of 1 demonstrated mild aqueous flare, mild vitreal haze, and aslightly obscured view of the retina. A score of 2 showed moderate aque-ous flare, dense vitreal haze, poor pupil dilation, and a moderately ob-scured view of the retina; and a score of 3 had intense aqueous flare,opaque vitreous, and a completely obscured view of the retina.

Histological analysis and quantification of retinal folding. Micewere sacrificed at 24, 48, 72, and 96 h following injection, and eyes wereenucleated using Stevens curved sharp-tip scissors. Eyes were fixed in 10%buffered formalin, embedded in paraffin, sectioned, and stained with he-matoxylin and eosin. To quantitate the extent of retinal folding in BALB/cand BALB(gld) mice, each section (5 or 6 sections per eye) was examinedby a blinded observer who counted the number of retinal folds per 10-�msection (5 or 6 sections per eye) (22, 23). These sections were cut throughor immediately adjacent to the optic nerve.

Quantification of intraocular bacteria. Mice were sacrificed at 24, 48,72, and 96 h following injection, and eyes were enucleated using Stevenscurved sharp-tip scissors. Eyes were placed in 1.0 ml sterile PBS on ice andhomogenized by bead beating (FastPrep FP120; Thermo Fisher Scientific,Waltham MA) with 1.0-mm glass beads for 45 s at a maximum speed.Homogenates were serially diluted and plated on brain heart infusion agarplates as previously described (21).

Electroretinography. Electroretinography was performed on mice at24, 48, 72, and 96 h postinjection using a slight modification of a previousprotocol (10). Briefly, mice were dark adapted for at least 4 h and wereanesthetized, and the pupils were dilated using 1% tropicamide ophthal-mic solution (Bausch & Lomb, Tampa, FL). Following anesthesia, bodytemperature was maintained at 37°C using a microwave heat pad. Goldwire electrodes (0.25 mm) (Alfa Aesar, Ward Hill, MA) were placed on thecornea after application of a hypromellose ophthalmic prism solution(Akorn, Inc.) and connected to a visual electrodiagnostic system (UTAS-E3000; LKC Technologies, Gaithersburg, MD). Needle electrodes placed inthe anterior scalp and the tail served as reference and ground leads, re-spectively. The b-wave amplitude (trough of a-wave to peak of b-wave) inresponse to a bright flash in a Ganzfeld illumination sphere was assessed inthe injected left eye and contralateral (internal control) right eye simulta-neously. A total of 30 readings taken at 0.6 cd-s/m2 flash intensity with a1-s interval between flashes were taken and averaged. The retinal functionwas defined as the ratio of the b-wave amplitudes of the experimentalinfected eye to the contralateral nontreated eye.

Myeloperoxidase assay. Mice were sacrificed at 24 and 48 h followinginjection, and eyes were enucleated using Stevens curved sharp-tip scis-sors. Eyes were snap-frozen in liquid nitrogen and were homogenized in1.0 ml of an assay buffer provided by an MPO fluorometric detection kit(Stressgen Assay Designs, Ann Arbor, MI). The homogenates were readusing a multidetection microplate reader (Synergy 2; BioTEK, HighlandPark, VT) and the concentration was derived from an MPO standardcurve, prepared as directed in the detection kit.

Statistical analysis. Where appropriate, the data were analyzed withan unpaired t test with P values of �0.05 as the basis for rejection of thenull hypothesis. The Gehan-Breslow-Wilcoxon and log rank (Mantel-Cox) tests were used to analyze the percentage of eyes destroyed followinginfection. Statistical analysis and graphing were performed on a computer(Prism, Gen5, and Microsoft Excel).

RESULTSEndophthalmitis in C57BL/6 and BALB/c mice. To determinewhether BALB/c mice are more resistant to S. aureus-induced en-dophthalmitis than C57BL/6 mice, wild-type BALB/c andC57BL/6 mice were injected intravitreally with an increasing con-

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centration of S. aureus (500, 2,500, 5,000, and 10,000 CFU of S.aureus) (see Fig. S1 in the supplemental material). Eyes were ex-amined by slit lamp microscopy and given a score of 0, 1, 2, or 3 (0,cleared; 3, destroyed). From these scores, the percentage of eyesdestroyed (score of 3) was calculated (see Fig. S1). Both strains ofmice efficiently cleared 500 CFU, while 10,000 CFU destroyed alleyes in both strains. However, a strain difference in resistance toendophthalmitis became apparent at the 2,500- and 5,000-CFUdose. At 2,500 CFU, 80% of C57BL/6 eyes were destroyed, com-pared to only 28% in the BALB/c group. Even at 5,000 CFU,the BALB/c mice exhibited increased resistance, where 90% of theC57BL/6 eyes were destroyed, compared to only 70% in theBALB/c group.

The 2,500-CFU dose, where the most significant difference inresistance was observed between strains, was chosen to furtherelucidate the strain difference in resistance to endophthalmitis.BALB/c and C57BL/6 mice were injected intravitreally with 2,500CFU of S. aureus and eyes were examined by slit lamp microscopyand given a score of 0, 1, 2, or 3 (0, cleared; 3, destroyed) (Fig. 1C).

From these scores, the percentage of eyes destroyed (score of 3)was calculated (Fig. 1D). In C57BL/6 mice, endophthalmitis de-veloped rapidly, with 28% of the eyes destroyed as early as 48 hpostinfection, compared to 0% in the BALB/c group. By 96 hpostinfection, 86% of the C57BL/6 eyes infected with 2,500 CFUof S. aureus were destroyed, compared to only 29% of the BALB/ceyes. A representative example of disease progression in C57BL/6versus BALB/c mice is shown macroscopically and histologicallyat 24, 48, 72, and 96 h postinfection (Fig. 1A and B). In BALB/cmice, slit lamp examination revealed mild inflammation thatpeaked at 48 h postinfection and subsequently resolved fully by 96h postinfection. In contrast, C57BL/6 mice displayed severe in-flammation, characterized by significant vitreal and aqueous hazeand poor pupil dilation at 48 h postinfection. The marked differ-ence in the percentage of eyes destroyed in C57BL/6 mice (86%)compared to that in BALB/c mice (29%) demonstrates increasedresistance to bacterial endophthalmitis in BALB/c mice.

Bacterial quantification. We performed studies to determinewhether C57BL/6 mice contained a higher bacterial load, indicat-

FIG 1 Clinical examination and retinal histology of C57BL/6J and BALB/cJ mice following intravitreal injections of 2,500 CFU of S. aureus. At 24, 48, 72, and96 h postinoculation, the eyes were examined by slit lamp microscopy. Representative eyes of BALB/c (A) and C57BL/6 (B) mice were photographed, enucleated,sectioned, and stained with H&E. (C) Each eye was given a score of 0, 1, 2, or 3 (0, cleared; 1, mild aqueous flare and vitreal haze; 2, moderate aqueous flare anddense vitreal haze; 3, destroyed). (D) At 96 h, 86% of eyes from C57BL/6 mice and 29% of eyes from BALB/c mice were destroyed (score, 3). H&E pictures weretaken of whole eyes at �4 magnification. n � 14 C57BL/6 mice, n � 15 BALB/c mice. *, P � 0.001 (Gehan-Breslow-Wilcoxon and log rank [Mantel-Cox] tests).

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ing an inability to effectively clear the infection. Bacterial quanti-fication was performed on whole eyes enucleated at 24, 48, and 96h postinfection (Fig. 2). No significant difference in bacterial loadwas detected between C57BL/6 and BALB/c mice at 24 h postin-fection. At 48 h postinfection, the number of viable bacteria re-

covered from C57BL/6 eyes was approximately 3 logs greater thanthat from BALB/c mice. By 96 h, bacterial load in C57BL/6 miceremained approximately 3 logs greater (maximum, 2.4 � 106

CFU/eye) than that in BALB/c (maximum, 6 � 103 CFU/eye).These data indicate that C57BL/6 mice are unable to effectivelyclear a 2,500-CFU S. aureus inoculum and are therefore moresusceptible to S. aureus-induced endophthalmitis.

Retinal function and histopathology. Retinal function, as de-termined by the b-wave amplitude measured by ERG, corre-sponded with the histological findings. Control mice that receivedsterile-saline intravitreal injections displayed a transient loss ofretinal function at 24 h postinjection, returning to 80 to 90% ofnormal by 96 h (Fig. 3A). BALB/c mice displayed a loss in retinalfunction that was approximately 40% of normal at 24 h postinfec-tion and recovered to approximately 50% of normal by 96 hpostinfection (Fig. 3A). In contrast, C57BL/6 displayed completeloss of retinal function at 24 h postinfection, with no recovery at 96h postinfection. Histological analysis at 24 h postinfection re-vealed a significant neutrophil infiltrate in both BALB/c andC57BL/6 mice (Fig. 3B), and myeloperoxidase analysis revealedno significant difference in the number of infiltrating neutrophils(7.1 � 0.2 [BALB/c] versus 7.4 � 0.2 [C57BL/6] relative units ofMPO; n � 10/group). At higher magnification, hematoxylin andeosin (H&E) staining of retinas from C57BL/6 mice reveals signif-

FIG 2 Bacterial quantification of C57BL/6 and BALB/c mice following intra-vitreal injections of 2,500 CFU of S. aureus. Bacterial quantification was per-formed at 24, 48, and 96 h postinoculum, and data are presented as CFU �standard errors of the means (SEM). The bacterial quantification revealed asignificantly increased bacterial load in C57BL/6 mice (n � 10) compared tothat of BALB/c mice (n � 10) at 48 and 96 h postinoculum. **, P � 0.001.

FIG 3 Retinal histology and ERG assessment of retinal function in C57BL/6 and BALB/c mice following intravitreal injections of sterile saline or 2,500 CFU ofS. aureus. (A) The b-wave amplitude was determined, and the data are presented as the ratio of injected eye to contralateral internal control eye � SEM. Thesaline-injected control groups displayed a transient loss of retinal function. In contrast, C57BL/6 mice lost complete retinal function in the injected eye, whileBALB/c mice displayed a loss in retinal function of 48% of normal. At 24 h (B) and 96 h (C), representative eyes were enucleated, sectioned, and stained with H&E.White arrows (neutrophils in the retina), black arrows (neutrophils along the RGC layer), black arrowheads (folds in RGC layer) (L, lens;V, vitreous; R, retina;RGC, retina ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer). H&E pictures were taken at �4 and �60 magnification at 24 h and �10magnification at 96 h. Saline, n � 3 BALB/c mice and n � 3 C57BL/6 mice; 2,500 CFU of S. aureus, n � 5 BALB/c mice and n � 5 C57BL/6 mice. **, P � 0001.

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icant neutrophil infiltration throughout the inner and outer plex-iform layers, correlating with substantial retinal damage (Fig. 3B).In contrast, in BALB/c mice, the neutrophils were primarily lo-cated in the vitreous and lining the retinal ganglion cell layer, withvery few neutrophils infiltrating only the inner plexiform layer(Fig. 3B). By 96 h postinfection, the inflammation subsided sig-nificantly in BALB/c mice (Fig. 3C). However, the prolonged lossof retinal function (50% of normal) in BALB/c mice correspondedwith the presence of retinal folding that was not observed inBALB/c mice injected with saline only (Fig. 3C). In contrast, his-tological analysis of eyes from C57BL/6 mice at 96 h postinfectiondemonstrated significant inflammation and complete destructionof the neural retina (Fig. 3C). Taken together, these data supportthe conclusion that C57BL/6 mice are more susceptible to S. au-reus-induced endophthalmitis than BALB/c mice.

Endophthalmitis in BALB(gld) mice. FasL is required for bac-terial clearance in C57BL/6 mice (10). BALB/c mice express apolymorphism of FasL that exhibits 9-fold-higher binding affinityto the Fas receptor and cytotoxic activity against Fas� target cellsthan the FasL expressed in C57BL/6 mice (17). The following se-ries of studies was performed to determine whether the relativeresistance to S. aureus-induced endophthalmitis in BALB/c miceis dependent upon Fas ligand. BALB(gld) mice expressing a mu-tant/nonfunctional form of FasL were challenged with an intrav-itreal injection of 2,500 CFU of S. aureus. No significant differencewas observed in the percentage of eyes destroyed, with 40% de-struction in BALB/c and 35% in BALB(gld) mice (Fig. 4A). More-over, there was no significant difference in bacterial load at 96 hpostinfection in BALB/c and BALB(gld) mice (Fig. 4B). Takentogether, these data demonstrate that the increased resistance toendophthalmitis in BALB/c mice does not depend on FasL and,further, that FasL is not required for bacterial clearance in BALB/cmice.

Retinal function in BALB/c and BALB(gld) mice. Interest-ingly, while there was no significant difference in bacterial clear-ance between BALB/c and BALB(gld) mice, there was a significantdifference in retinal function retained at 96 h postinfection. Inorder to assess retinal function in mice that cleared the infection,ERG was performed only on eyes with a clinical score of �3. Inmice that successfully cleared the infection, BALB/c mice retainednearly 58% of normal retinal function at 96 h postinfection,compared to only 36% of normal retinal function retained in

BALB(gld) mice (Fig. 5A). Histological analysis at 96 h postinfec-tion revealed extensive retinal folding in BALB(gld) retinas com-pared to BALB/c retinas (Fig. 5C and D). Quantification of retinalfolds in histological sections taken from eyes at 96 h postinfectionfurther corroborates the ERG results, demonstrating a significantincrease in retinal folding in BALB(gld) compared to BALB/c mice(Fig. 5B). These data imply that while FasL is not required forbacterial clearance, it is critical for protecting host tissue frominflammation-induced damage.

Neutrophil infiltration in BALB/c and BALB(gld) mice. Neu-trophils are the predominant cell type present in the early innateimmune-mediated inflammatory response to ocular infections (8,9, 24). A delay in neutrophil infiltration will allow the bacteria tomultiply, leading to the development of destructive endophthal-mitis. On the other hand, a prolonged neutrophil presence canlead to nonspecific host tissue destruction. The constitutive ex-pression of Fas ligand in the eye is thought to protect ocular tissuefrom the damaging effects of inflammation by inducing apoptosisof Fas�-infiltrating inflammatory cells and preventing inflamma-tion (13, 25). Therefore, we performed a myeloperoxidase assay toquantify the neutrophil infiltrate and to determine if the increasedretinal damage observed in the BALB(gld) mice was due to a pro-longed neutrophil infiltrate (Fig. 6). The myeloperoxidase analysisrevealed no significant difference in the number of neutrophils at24 h postinfection, indicating no delay in neutrophil infiltration inBALB(gld) mice. However, while the number of neutrophils sig-nificantly decreased in WT mice at 48 h postinfection, the num-bers of neutrophils remained elevated and actually increased inthe absence of functional FasL in BALB(gld) mice at 48 h postin-fection. This prolonged neutrophil infiltration correlates well withthe increased retinal damage observed in BALB(gld) mice (Fig.5A). These data indicate that in the absence of functional FasL,there is prolonged neutrophil presence that leads to increasednonspecific tissue damage in BALB(gld) mice.

DISCUSSION

It was demonstrated previously that FasL was required for bacte-rial clearance from the posterior segment of C57BL/6 mice chal-lenged with S. aureus (10). Herein, we demonstrate that BALB/cmice that express an allotypic polymorphism of FasL resulting in a9-fold increase in cytotoxic activity are more resistant thanC57BL/6 mice to S. aureus-induced endophthalmitis. An intrav-

FIG 4 Clinical examination and bacterial quantification of BALB/c and BALB(gld) mice following intravitreal injections of 2,500 CFU of S. aureus. At 24, 48, 72,and 96 h postinoculum, the eyes were examined by slit lamp microscopy. (A) Each eye was given a score of 0, 1, 2, or 3 (0, cleared; 1, mild aqueous flare and vitrealhaze; 2, moderate aqueous flare and dense vitreal haze; 3, destroyed). There was no significant difference in percentage of eyes destroyed. At 96 h postinoculum,40% of the eyes were destroyed (score, 3) in BALB/c and 35% in BALB(gld) mice. BALB/c mice, n � 10; BALB(gld) mice, n � 10. (B) Bacterial quantificationrevealed no significant difference in bacterial load at 96 h postinoculum between BALB/c mice (n � 15) and BALB(gld) mice (n � 15).

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itreal injection of 2,500 CFU of S. aureus resulted in the destruc-tion of 86% of the eyes in C57BL/6 mice, compared to only 29%destruction in BALB/c mice. To our surprise, though, the in-creased resistance in BALB/c mice was not dependent upon FasL.BALB(gld) mice were able to clear an S. aureus infection as effi-ciently as BALB/c mice. However, while the absence of FasL didnot impede bacterial clearance in BALB(gld) mice, increased ret-inal damage and loss of retinal function was observed followingbacterial clearance in BALB(gld) mice compared to in BALB/c

mice. Moreover, the increased retinal damage in BALB(gld) micecoincided with a prolonged neutrophil infiltration. Taken to-gether, these data demonstrate that in BALB/c mice, FasL is notrequired for bacterial clearance, but, rather, FasL is critical in res-olution of the inflammatory response and protecting the retinafrom nonspecific damage.

FasL is constitutively expressed on ocular tissue, where it isthought to be critical in the maintenance of immune privilege byinducing apoptosis of Fas�-infiltrating inflammatory cells (13,25). While FasL was first identified as an apoptosis-inducing li-gand, more recent studies revealed nonapoptotic functions ofFasL, where FasL triggered the Fas receptor to promote cell pro-liferation, migration, and release of proinflammatory cytokines(26–31). While it is not completely understood how the differentpathways are regulated in vivo, it is thought that the Fas receptorengages these pathways in a cell- and tissue-dependent manner.Moreover, Gao and colleagues suggested a link between FasL-me-diated apoptosis and immune deviation in the eye that is depen-dent upon the microenvironment, in particular, the production ofTh2 versus Th1 cytokines (32). Promotion of Th2-type cytokines(interleukin 10 [IL-10]) inhibits inflammation and minimizes theinflammatory-induced damage to the ocular tissue. Many studieshave documented that C57BL/6 mice favor a Th1 response toinfection, while, BALB/c mice favor a Th2-like response, and theseresponses are characterized by differences in the cytokines pro-duced: Th1-like responses are characterized by the production ofIL-2 and gamma interferon (IFN-y), and Th2-like responses arecharacterized by the production of IL-3, IL-4, IL-5, IL-6, andIL-10 (33–35). Therefore, these strain-specific differences in the

FIG 5 Retinal histology and ERG assessment of retinal function of BALB/c and BALB(gld) mice following intravitreal injections of 2,500 CFU of S. aureus. (A)The b-wave amplitude was determined, and the ratio of injected eye to contralateral internal control eye was calculated. The BALB/c mice lost 42% of normalretinal function in the infected eye, while BALB(gld) mice lost nearly 64%. (B) At 96 h, representative eyes were enucleated, sectioned, and stained with H&E. Mildinflammation was observed in BALB/c mice, while BALB(gld) mice presented with significant retinal damage. INL, inner nuclear layer; ONL, outer nuclear layer.(C and D) H&E pictures were taken at �4 magnification and �20 magnification. BALB/c mice, n � 10; BALB(gld) mice, n � 10. *, P � 0.05; **, P � 0.01.

FIG 6 PMN infiltration into infected eyes of BALB/c and BALB(gld) micefollowing intravitreal injections of 2,500 CFU of S. aureus. At 24 and 48 hpostinoculum, the eyes were enucleated and processed for myeloperoxidase(MPO). Similar MPO activity was observed in BALB/c and BALB(gld) mice at24 h postinoculum. At 48 h, persistent PMN infiltration was observed inBALB(gld) compared to the significant decrease in PMN infiltration observedin BALB/c mice. BALB/c mice, n � 5; BALB(gld) mice, n � 5. *, P � 0.001.

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microenvironment during an infection may alter FasL functionand ultimately susceptibility to S. aureus endophthalmitis.

In 2005, Engelbert and Gilmore demonstrated that FasL wasrequired for the early recruitment of granulocytes that are criticalmediators of bacterial clearance in S. aureus endophthalmitis inC57BL/6 mice (10). In C57BL/6(gld) mice (that express a non-functional FasL), the inability to clear an S. aureus infection cor-responded with a significant decrease in the number of infiltratinggranulocytes at 24 h postinfection. This implied that increasedsusceptibility to infection coincided with decreased neutrophil in-filtration, while increased resistance to bacterial infection, as weobserved in BALB/c mice, would coincide with a quicker and/ormore robust neutrophil infiltration. To our surprise, we observedno difference in the rate or quantity of neutrophil infiltration be-tween the more resistant BALB/c and more susceptible C57BL/6strains of mice at 24 h postinfection (7.1 � 0.2 versus 7.4 � 0.2relative units of MPO; n � 10/group). In addition, there was noreduction in the number of infiltrating neutrophils in BALB(gld)mice that express a nonfunctional FasL compared to BALB/cmice. Taken together, these data indicate that in contrast toC57BL/6 mice, FasL is not required for the infiltration of neutro-phils in BALB/c mice. Moreover, the increased resistance ofBALB/c mice to S. aureus infection (only 29% of BALB/c eyesinfected with 2,500 CFU of S. aureus were destroyed compared to86% of the C57BL/6 eyes) is not dependent upon the rate and/ornumber of neutrophils that infiltrate the eye.

Many studies have documented several differences in the im-mune response of C57BL/6 and BALB/c mice. Studies comparingthe host immune response between resistant and susceptiblestrains of mice have been performed extensively to dissect themechanisms of innate and adaptive immunity critical to resistancein several mouse models of infection, including bacterial keratitis(34–38). In regard to differences in immune mechanisms, Hazlettand colleagues established an important role for adaptive immu-nity in bacterial keratitis, demonstrating that CD4� T cells con-tribute to the pathogenesis of P. aeruginosa keratitis in C57BL/6mice (34, 39). Moreover, mouse strains favoring the developmentof a Th1-type response (C57BL/6) are susceptible, while mice fa-voring a Th2-type response (BALB/c) are resistant. Interestingly,though, while an important role for adaptive immunity has beenestablished in bacterial keratitis, such a role for adaptive immunityin endophthalmitis has not yet been demonstrated. While it is welldocumented that the anterior segment displays significantly in-creased resistance to infection compared to the posterior segment(40, 41), no studies have yet been performed to examine the role ofTh1- and Th2-type responses in resistance to infection in the pos-terior segment. While our data demonstrate increased resistanceto S. aureus endophthalmitis in BALB/c mice that favor a Th2-typeresponse, additional studies will be required to fully elucidate themechanisms critical to resistance to infection in the posterior seg-ment.

Unlike the adaptive immune response, the innate immune re-sponse is immediate and has been shown to be critical in bacterialclearance during endophthalmitis (10). Recent studies have alsobegun to investigate the effect the microenvironment has on thedevelopment of the innate immune response, in particular neu-trophil activation (42). Therefore, it is possible that while our datashow no difference in the rate of neutrophil infiltration betweenresistant and susceptible mouse strains, the neutrophil functionand ability to clear the bacteria may be different between strains of

mice. This possibility is supported by a study of resistance andsusceptibility to methicillin-resistant S. aureus (MRSA), wheretwo different subsets of neutrophils were identified in mice withdifferent susceptibilities to infection by MRSA (43). One subset ofneutrophils produced IL-12 and CCL3 and was associated withresistance to MRSA, while a second subset of neutrophils pro-duced IL-10 and CCL2 and was associated with susceptibility toMRSA (43). In addition, a recent study using a subcutaneousStaphylococcus aureus infection model demonstrates an associa-tion between increased resistance with neutrophil infiltration anda Th2-like response (44). However, an alternate possibility is thatneutrophils are not the critical factor in the increased resistance toS. aureus endophthalmitis observed in BALB/c mice, but ratheranother component of innate immunity, such as antimicrobialfactors, is the critical factor. Future studies will be focused onfurther elucidating the immune mechanism(s) responsible for theincreased resistance to S. aureus endophthalmitis in BALB/c mice.

In BALB/c mice, the function of FasL appears to be moreclosely associated with the prevention of nonspecific tissue dam-age secondary to host-mediated inflammation. This is furthersupported by the prolonged neutrophil infiltration, increased in-filtration into the retina, and increased retinal damage observed inBALB(gld) mice that express a nonfunctional form of FasL. Previ-ous studies by our laboratory and others demonstrated that withinthe retina, FasL is primarily expressed on retinal microglia, astro-cytes, the inner nuclear layer, and the retinal pigment epithelium(45–47). Therefore, we would predict that microglia within theretina ganglion cell (RGC) layer and astrocytes that line the retinaare most likely to play a critical role in inducing apoptosis of in-filtrating neutrophils, resulting in reduced infiltration of neutro-phil into the retina and shutting down the innate-mediated in-flammation. A previous study using a corneal keratitis modeldemonstrates that FasL is required for the clearance of infiltratingneutrophils and resolution of inflammation (48). In the absenceof FasL, polymorphonuclear leukocyte (PMNs) persisted, result-ing in increased nonspecific corneal damage. Moreover, othersdemonstrate that FasL.2 (expressed in BALB/c mice) is muchmore efficient at killing Fas� targets than FasL.1 (expressed inC57BL/6 mice) (17). Taken together, these data suggest that thesharp decline in PMNs we observe at 48 h postinfection in BALB/cmice is due to the expression of FasL, in particular FasL.2, which ismore cytotoxic for Fas� targets and thus more efficient at killingFas� neutrophils. However, additional studies must be performedto specifically identify the FasL� cells critical in inducing apopto-sis of Fas� inflammatory cells.

In conclusion, our data demonstrate increased resistance to S.aureus endophthalmitis in BALB/c versus C57BL/6 mice via anFasL-independent mechanism. In addition, the fact that (i) FasL isrequired for bacterial clearance in C57BL/6 mice but not BALB/cmice and (ii) FasL is critical in protecting the retina from nonspe-cific damage in BALB/c but not C57BL/6 mice reveals the com-plexity of the Fas-FasL system and how the function of FasL inocular innate immunity may be differentially regulated by geneticbackground. Specifically, differences in genetic background mayalter FasL function through changes in the immune response phe-notype (Th1 versus Th2), leading to changes in (i) the cytokinemilieu, (ii) the form of FasL expressed, and (iii) the susceptibilityof Fas� targets to apoptosis. Therefore, the diverse activities ofFasL and the complexity of the Fas-FasL system must be taken into

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account when elucidating the function of FasL in a particular dis-ease model.

ACKNOWLEDGMENTS

We thank Marie Ortega and Santina Caruso for their excellent assistancewith animal surgical procedures and animal breeding. We also thank Par-ticia D’Amore for helpful comments and assistance with editing the man-uscript.

This work was supported by the National Institutes of Health RO1-EY016145 (to M. S. Gregory) and the Department of Defense W81XWH-07-2-0038 (to M. S. Gregory).

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