-Independent Pathways by Signaling through MyD88-Dependent

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-Independent Pathwaysby Signaling through MyD88-Dependent and

KeratitisPseudomonas aeruginosaRegulate TLR4 and TLR5 on Corneal Macrophages

PearlmanYiping Han, Charles M. Stopford, Arne Rietsch and Eric

Shive,Ramadan, Susan R. Williams, Scott Howell, Carey L. Yan Sun, Mausita Karmakar, Sanhita Roy, Raniyah T.

http://www.jimmunol.org/content/185/7/4272doi: 10.4049/jimmunol.1000874September 2010;

2010; 185:4272-4283; Prepublished online 8J Immunol

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The Journal of Immunology

TLR4 and TLR5 on Corneal Macrophages RegulatePseudomonas aeruginosa Keratitis by Signaling throughMyD88-Dependent and -Independent Pathways

Yan Sun,* Mausita Karmakar,*,† Sanhita Roy,* Raniyah T. Ramadan,* Susan R. Williams,*

Scott Howell,* Carey L. Shive,† Yiping Han,‡ Charles M. Stopford,x Arne Rietsch,x and

Eric Pearlman*,†

Pseudomonas aeruginosa is a major cause of blindness and visual impairment in the United States and worldwide. Using a murine

model of keratitis in which abraded corneas are infected with P. aeruginosa parent and ΔfliC (aflagellar) strains 19660 and PAO1,

we found that F4/80+ macrophages were the predominant cell type in the cornea expressing TLR2, TLR4, and TLR5. Depletion of

macrophages and dendritic cells using transgenic Mafia mice, in which Fas ligand is selectively activated in these cells, resulted in

diminished cytokine production and cellular infiltration to the corneal stroma and unimpaired bacterial growth. TLR42/2 mice

showed a similar phenotype postinfection with ΔfliC strains, whereas TLR4/52/2 mice were susceptible to corneal infection with

parent strains. Bone marrow-derived macrophages stimulated with ΔfliC bacteria induced Toll/IL-1R intracellular domain (TIR)-

containing adaptor inducing IFN-b (TRIF)-dependent phosphorylation of IFN regulatory factor 3 in addition to TIR-containing

adaptor protein/MyD88-dependent phosphorylation of IkB and nuclear translocation of the p65 subunit of NFkB. Furthermore,

TRIF2/2 mice showed a similar phenotype as TLR42/2 mice in regulating only ΔfliC bacteria, whereas MyD882/2 mice were

unable to clear parent or ΔfliC bacteria. Finally, IL-1R12/2 and IL-1a/b2/2 mice were highly susceptible to infection. Taken

together, these findings indicate that P. aeruginosa activates TLR4/5 on resident corneal macrophages, which signal through TRIF

and TIR-containing adaptor protein/MyD88 pathways, leading to NF-kB translocation to the nucleus, transcription of CXCL1

and other CXC chemokines, recruitment of neutrophils to the corneal stroma, and subsequent bacterial killing and tissue damage.

IL-1a and IL-1b are also produced, which activate an IL-1R1/MyD88-positive feedback loop in macrophages and IL-1R on other

resident cells in the cornea. The Journal of Immunology, 2010, 185: 4272–4283.

Pseudomonas aeruginosa is an important human pathogen,causing severe pulmonary disease, especially in patientswith cystic fibrosis. However, P. aeruginosa is also a global

cause of blindness and visual impairment. The major risk factors forP. aeruginosa keratitis include traumatic injury, pre-existing cornealabnormalities, and contact lens wear. As there are an estimated 140million contact lens wearers worldwide, P. aeruginosa keratitis hasan important clinical and economic impact (1–3). Disruption of theprotective corneal epithelium facilitates P. aeruginosa invasion intothe corneal stroma, although there is evidence that these organisms

can penetrate the corneal epithelium (4). Once in the stroma, bac-terial replication and the host response both contribute to loss ofcorneal transparency and development of corneal opacification. Inaddition, the P. aeruginosa Type III Secretion System has a role incorneal disease (5–7).Studies in our laboratory demonstrated that TLR activation of

the abraded corneal surface results in rapid chemokine production,neutrophil and macrophage recruitment to the corneal stroma, anddevelopment of corneal haze (8–10). We also demonstrated thatadaptor molecules MyD88 and Toll/IL-1R intracellular domain(TIR)-containing adaptor inducing IFN-b (TRIF) are functional inthe cornea and that MyD88 has a novel inhibitory role in TLR3/TRIF activation (8, 9, 11). Although we and others reported thatcorneal epithelial cells respond to TLR ligands (9, 12–17), weshowed that LPS/TLR4-induced corneal inflammation is also reg-ulated by macrophages and dendritic cells (18).In the current study, we examined the role of macrophages and

TLRs in a murine model of P. aeruginosa keratitis using parent (fla-gellar) and nonflagellar ΔfliC mutants of two P. aeruginosa strainsdescribed as having either cytotoxic (19660) or invasive (PAO1)properties in the cornea (19). Our findings indicate that activation ofTLR4 and TLR5 on corneal macrophages rapidly stimulates pro-duction of proinflammatory and chemotactic cytokines and cellularinfiltration to the corneal stroma, which limit bacterial growth andsurvival. We also show that P. aeruginosa activates NF-kB in mac-rophages through both the TIR-containing adaptor protein (TIRAP)/MyD88 dependent and the TRIF-dependent pathways, and thatIL-1a and IL-1b activation of IL-1R1 is an important feedbackpathway in the host response to P. aeruginosa.

*Department of Ophthalmology and Visual Sciences, †Department of Pathology,‡Department of Periodontics, and xDepartment of Molecular Biology and Microbi-ology, Case Western Reserve University, Cleveland, OH 44106

Received for publication March 18, 2010. Accepted for publication July 23, 2010.

This work was supported by National Institutes of Health Grants R01 EY14362(to E.P.), P30 EY11373 (to E.P.), and DE14924 (to Y.H.) and American CancerSociety Research Scholar Grant RSG-09-198-01-MPC (to A.R.). Additional supportfor this work was provided by the Research to Prevent Blindness Foundation and theOhio Lions Eye Research Foundation.

Address correspondence and reprint requests to Dr. Eric Pearlman, Department ofOphthalmology and Visual Sciences, Case Western Reserve University, 10900 EuclidAvenue, Cleveland, OH 44106. E-mail address: [email protected]

Abbreviations used in this paper: 7-AAD, 7-aminoactinomycin D; ac, anterior cham-ber; epi, corneal epithelium; Fla, flagellin; Inoc, inoculum; IRF3, IFN regulatoryfactor 3; MD-2, myeloid differentiation factor-2; MIF, migratory inhibition factor;MPO, myeloperoxidase; P, phosphorylated; TIR, Toll/IL-R intracellular domain;TIRAP, Toll/IL-R intracellular domain-containing adaptor protein; TRAM, Toll/IL-1R intracellular domain-containing adaptor-inducing IFN-b–related adaptor mole-cule; TRIF, Toll/IL-R intracellular domain-containing adaptor inducing IFN-b.

Copyright� 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

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Materials and MethodsP. aeruginosa strains and preparation

P. aeruginosa strain 19660 was obtained from American Type CultureCollection (Manassas, VA), whereas strain PAO1 was maintained as stock.The fliC deletion constructs for both strains were generated by amplifying∼500-bp flanking regions using primers fliC5-1 (59-AAAAATCTAGA-CGCCGCGGCCGGCGCGCAGTT-39), fliC5-2 (59-AACTCGAGCCGCA-AGCATGCTGAAGGCCATGGTGATTTCCT CCAAAG-39), fliC3-1 (59-TTCAGCATGCTTGCGGCTCGAGTTCGCTAAGCCCGGGAA CGGTC-ACT-39), and fliC3-2 (59-AAAAAAAGCTTCAAGCACCATGC CAAGTC-GTTCAA-39). The two flanking regions were spliced together by overlapextension PCR and cloned into the allelic exchange vector pEXG2 as anXbaI-HindIII fragment (20, 21). The vector was then delivered intoP. aeruginosa bymating and generating a cointegrate, which was resolved by sucrose selection.Mutants that had retained the deletion were identified by loss of motility andconfirmed by PCR. The complementing plasmid pP40-fliCpwas generated byamplifying the fliC open reading frame and promoter region using primers fliCp-5R (59-AAAAAGAATTCGCGTGCCCTGTTGCACGGG AGGGCT-39)and fliC3H (59-AAAAAAGCTTCGGGCTTAGCGCAGCAGGCTCAGGA-39) and cloning the resulting PCR fragment into the promoterless shuttle-vector pPSV40 as an EcoRI/HindIII fragment (20).

For infection studies, bacteria were grown in brain-heart infusion broth(BD Diagnostics, Sparks, MD) at 37˚C for 18 h, and log-phase subculturewas grown to an optic density of 0.200 (OD 650 nm, ∼1 3 108 CFU/ml).Bacteria were washed three times in PBS (HyClone, Logan, UT), centri-fuged at 3000 rpm for 5 min, and resuspended in PBS. Dilution of thesample was made with PBS for the final inoculum. CFUs were quantifiedby plating track dilutions of the inoculum onto brain-heart infusion agar.

Source of mice

C57BL/6,IL-1R12/2 mice and BALB/c and C.C3-TLR4Lps-d/J mice ona BALB/c background were purchased from The Jackson Laboratory (BarHarbor, ME). TLR42/2, MyD882/2, and TRIF2/2 mice were obtainedwith permission from Dr. S. Akira (Research Institute for Microbial Dis-eases, Osaka University, Japan), TIRAP2/2 mice were obtained from Dr.R. Medzhitov, and TLR52/2 mice were obtained from Dr. Richard Flavell(both from the Howard Hughes Medical Institute, Yale University, NewHaven, CT). Myeloid differentiation factor-2 (MD-2)2/2 mice fromK. Miyaki and IL-1a, b, and IL-1a/b mice were obtained fromDr. Y. Iwakura (both from the University of Tokyo, Tokyo, Japan). TLR4/52/2 mice were generated at Case Western Reserve University AnimalFacility (Cleveland, OH), and Mafia mice were obtained from Dr. S.Burnett (University of Utah, Provo, UT). All animals were housed underspecific pathogen-free conditions in microisolator cages and maintainedaccording to institutional guidelines and the Association for Research inVision and Ophthalmology Statement for the Use of Animals in Oph-thalmic and Vision Research.

Mafia mouse model for depletion of macrophages anddendritic cells

Transgenic mice were generated by Burnett et al. (22) for induciblemacrophage and dendritic cell depletion by macrophage Fas-induced ap-optosis. The transgene is under control of the c-fms promoter that regulatesexpression of the CSF-1 receptor that is expressed on macrophages anddendritic cells. Mafia mice express eGFP and a membrane-bound suicideprotein made up of the human low-affinity nerve growth factor receptor,the FK506-binding protein, and a cytoplasmic domain of Fas (22, 23).AP20187 is a covalently linked dimer (Ariad Pharmaceuticals, Cambridge,MA) that cross-links the FK506-binding protein region of the suicideprotein and induces caspase 8-dependent apoptosis as described pre-viously. These mice are on a C57BL/6 background and have a normalphenotype in the absence of the dimerizer.

Bone marrow chimeric mice

Cells from C57BL/6TgN (ACTbEGFP)10sb (eGFP) mice or eGFP micecrossed with TLR42/2 were isolated as described previously (24). Fol-lowing euthanasia by CO2 asphyxiation, femurs and tibias were removed,and the shafts were centrifuged at 10,000 rpm for 30 s at 4˚C. The pelletwas resuspended in 1 ml sterile RBC lysis buffer for 2–3 min, and cellswere centrifuged at 1200 rpm for 5 min at room temperature and thenwashed once using sterile DMEM. Recipient mice received 2 3 600-Gydoses of whole-body irradiation 3 h apart. Immediately following thesecond dose, mice were injected i.v. with 200 ml DMEM containing 5 3106 bone marrow cells. The presence of eGFP+ cells in the blood and in thecornea indicated successful transplant.

In vivo model of corneal infection

Mice were anesthetized by i.p. injection of 0.4 ml 2,2,2-tribromoethanol(1.2%). Central corneas were scarified with three parallel 1 mm in lengthabrasions using a 26-gauge needle. A 5-ml aliquot containing ∼5 ml bac-terial suspension was applied to the scarified cornea. Sterile PBS wasapplied to the abraded cornea as a trauma control. A sterile trephine(Miltex, Tuttlingen, Germany) was used to generate a 2-mm-diameterpunch of silicone hydrogel contact lens (Night and Day; CIBA Vision,Duluth, GA), which was placed over the central cornea to maintainplacement of the bacterial suspension. The contact lenses were removedafter 2 h, and mice were allowed to recover from the anesthesia.

Quantification of live bacteria in infected corneas

Whole eyes were homogenized under sterile conditions using the MixerMill MM300 (Retsch, Newtown, PA) at 33 Hz for 4 min. Serial log dilutionswere performed and plated onto Brain-Heart Infusion Agar (BD Diag-nostics). Plates were incubated at 37˚C for 18 h, and the number of CFUswas determined by direct counting.

In vivo confocal microscopy

In vivo analysis of the corneal infiltration was evaluated using the Confo-Scan3 microscope system (Nidek Technologies, Fremont, CA) as describedin our previous studies (8, 25). Transparent gel (Genteal; Novartis Oph-thalmics, Duluth, GA) was applied at the interface of the cornea andobjective, and corneas were examined using a340 objective. The software(NAVIS; Lucent Technologies, Murray Hill, NJ) was used to capture se-quential images of the entire cornea.

Histology and immunohistochemistry

Eyes from control and infected mice were enucleated and placed in 10%formalin/PBS 5 mm corneal sections were prepared, and H&E staining wasperformed usingGill’s hematoxylin for 3–5min following deparaffinization.The slides were dipped into glacial acetic acid solution, placed in ShandonBluing Solution (Thermo Shandon, Pittsburgh, PA), and counterstainedwith alcohol Eosin Y solution (Thermo Shandon). The slides were thendehydrated and mounted under Permount medium (Vector Laboratories,Burlingame, CA).

To detect neutrophils in the corneal stroma after stimulation with LPS orflagellin, eyes were snap frozen, and 5-mm sections were incubated withprimary rat anti-mouse neutrophil (NIMP-R14; Abcam, Cambridge, MA)as described previously (25). Briefly, Abs were diluted 1/100 in 1% FCS-TBS 2 h at room temperature. Sections were washed and incubated withFITC-conjugated rabbit anti-rat Ab (Vector Laboratories) diluted 1/200in 1% FCS-TBS for 45 min. Neutrophils in the corneal stroma wereenumerated directly using fluorescence microscopy (magnification 340)(Olympus Optical, Tokyo, Japan).

Myeloperoxidase assay for neutrophils

Corneas were dissected and homogenized in tissue lyser in 200 ml/cornea0.5% hydratropic acid-Br buffer and 10 mM N-ethylmaleimide (Sigma-Aldrich, St. Louis, MO). The reaction mixture was prepared according tomanufacturer’s directions (Cell Technology, Mountain View, CA), andsamples were assayed along with myeloperoxidase (MPO) standards. After30 min of incubation in the dark, reactivity was measured using a fluo-rescent plate reader, and values were calculated as units of MPO activity.

Detection of cytokines in the cornea

Corneas were excised using a 2-mm trephine, residual iris material wasremoved, and corneas were homogenized using aMixerMillMM300 (Retsch,Haan, Germany) for 4 min at 30 Hertz/s. After centrifugation, cytokine levelsin supernatants were determined by sandwich ELISA, according to manu-facturer’s directions (R&D Systems, Minneapolis, MN). Absorption wasmeasured at 450 nm on a VersaMax microplate reader using using Soft-MaxPro software 5.2 (Molecular Devices, Sunnyvale, CA).

Flow cytometric analysis of corneas

Corneas were excised, and residual iris material was removed as describedabove. Five corneas were pooled, minced using a scalpel, and incubatedin 500 ml collagenase type I (Sigma-Aldrich) at ∼82 U/cornea for 1.5 h at37˚C. Cell suspensions were filtered and washed with 3 ml FACS buffer(13 PBS without Ca2+ or Mg2+, 1% FBS, and 0.1% sodium azide). Cellswere centrifuged and resuspended in 2 ml FACS buffer containing 20 mgFc blocking Ab (anti-mouse CD16/32; eBioscience, San Diego, CA) and

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incubated on ice for 10 min. After blocking, the corneal cells were thendistributed into 100 ml samples and stained with the following Abs: neu-trophil marker NIMP-14-Alexa Fluor 488 (in house, 0.5 mg/106 cells),macrophage marker CD11b-PE (1 mg/106 cells; eBioscience), F4/80-PE-Cy5 (0.3 mg/106 cells; eBioscience), dendritic cell marker CD11c-efluor TM450 (0.5 mg/106 cells; eBioscience), TLR2-Alexa Fluor 647(0.5mg/106 cells; eBioscience), TLR4-allophycocyanin (0.5 mg/106 cells;eBioscience), and TLR5-FITC (1 mg/sample; Abcam, Cambridge, MA); orwith isotype controls efluorTM450-Armenian Hamster IgG (0.5 mg/sample;eBioscience), PE-rat IgG2b (1 mg/sample; eBioscience), allophycocyanin-rat IgG2a (0.5 mg/sample; R&D Systems), and Alexa Fluor 647-rat IgG2b(0.5 mg/sample; eBioscience). Each cell type was also stained with thenucleic acid dye 7-aminoactinomycin D (7-AAD) (eBioscience) to accesscell viability, because dead or dying cells with a compromised plasmamembrane cannot exclude 7-AAD. Furthermore, 7-AAD fluoresces in thefar-red range of the spectrum and can be used with PE-stained cells withoutspectral overlap. Cells were stained for 30 min on ice and then washed with4 ml FACS buffer and resuspended in 0.5% formaldehyde. Cell profileswere acquired on a BD LSRII flow cytometer the following day. Cells weregated on forward versus side scatter, then cells positive for cell type markers(F4/80, NIMP-14, CD11b, and CD11c) were gated based on isotype markeror unstained cell sample. Gates were set either using the histogram or dot-plot graph, depending on which representation showed clear positive results.The percentage of cells positive for TLR5, TLR2, or TLR4 was then cal-culated for each cell type using the population hierarchy. Flow cytometricanalysis was performed using BD FACSDiva software.

Western blot and p65 translocation to the nucleus in bonemarrow derived macrophages

Bone marrow cells were derived from 6- to 8-wk-old C57BL/6 mice asdescribed above for development of chimeras. Cells were incubated inDMEM containing 10% FBS and 30% L929-conditioned medium for 10 d todifferentiate them to macrophages (9). These cells were then counted andincubated and stimulated with either LPS (100 ng/ml), or P. aeruginosa19660 parent strain or ΔfliC at a multiplicity of 100 for the indicated times.The bacteria used for stimulation were killed in 0.3% tobramycin solution asdescribed previously (25). After treatment, the cells were washed in ice-coldPBS and lysed in 13 lysis buffer (Cell Signaling Technology, Beverly, MA).Protein was quantified using standard BCA technique, and equal quantity ofprotein was fractionated in 10% SDS polyacrylamide gel. The protein wasthen transferred onto nitrocellulose membrane and probed using the fol-lowing primary Abs: phosphorylated (P)-IkBa, P-IFN regulatory factor 3(IRF3), total IkBa, and b-actin (Cell Signaling Technology) in 1/1000dilutions in 5% BSA. HRP-conjugated secondary Ab was used and de-veloped with Pierce SuperSignal Femto ECL.

To detect NF-kB nuclear translocation, 5 3 104 bone marrow-derivedmacrophages were cultured on sterile coverslips in 24-well plates and trea-ted with LPS (100 ng/ml), 19660 (multiplicity of infection 100) or19660DfliC (multiplicity of infection 100) for 15, 30, and 60 min. Followingactivation, the cells were fixed with 4% paraformaldehyde for 15 min atroom temperature. Fixed cells were permeabilized with 0.1% Triton X-100in PBS for 1 min at room temperature and incubated with rabbit anti-mousep65 (1/100; eBioscience) in PBS containing 10% goat serum for 1 h at roomtemperature. Coverslips were washed twice with PBS and incubated withAlexa Fluor 488-labeled goat anti-rabbit IgG Ab (Molecular Probes) in PBSat room temperature for 1 h and washed with PBS, and placed on glass slidesusing Vectashield mounting medium with DAPI (Vector Laboratories U.K.,Peterborough, U.K.). Images were captured on a Leica DMI 6000 B invertedmicroscope using a 340 objective connected to a Retiga EXI camera (Q-imaging, Vancouver, British Columbia, Canada).

Statistical analysis

Student t test or ANOVA with Tukey post hoc analysis (GraphPad Prism,San Diego, CA), and statistical significance was defined as p , 0.05.

ResultsTLRs are expressed on corneal macrophages, and depletion ofmacrophages and dendritic cells impairs bacterial killing

To determine which cells in the cornea express TLR2, TLR4, andTLR5 during infection with P. aeruginosa, corneas of C57BL/6mice were abraded, 13 105 CFU 19660 were added topically, anda 2-mm-diameter punch from a silicone hydrogel contact lens wasplaced on the corneal surface for 2 h to maintain contact between

the bacteria and the ocular surface. Corneas were dissected andhomogenized 18 h postinfection, and a single-cell suspension wasincubated with fluorescent-tagged F4/80 (macrophages) or withAbs to CD11c (dendritic cells) or NIMP R14 (neutrophils). Cellswere also incubated with Abs to TLR2, TLR4, or TLR5, or withisotype controls, and examined by flow cytometry. Dead cells wereidentified using 7-AAD and were gated out of the population, andpositive cell populations were gated according to the specific iso-type control. In a representative experiment (Fig. 1A), 71.2% F4/80+

cells expressed TLR2, 47.8% expressed surface TLR4, and 87.5%expressed TLR5. In contrast,,10%NIMPR14+ neutrophils expres-sed TLR2, TLR4, or TLR5, and as,4% of total cells were CD11c+,TLR expression could not be assessed (data not shown). We alsofound that CD11b Abs stained the same population of cells asF4/80+ macrophages (data not shown) and that there was an addi-tional population of TLR-expressing cells that were F4/80 negative(Fig. 1A) andwere also negative for CD11b, CD11c, andNIMP-R14(data not shown). These cell populations might include residentcorneal fibroblasts or epithelial cells. Overall, these data show thatF4/80+ macrophages are the predominant cell population that ex-presses TLR2, TLR4, and TLR5 in the cornea during P. aeruginosakeratitis.We recently reported that LPS-induced corneal inflammation is

dependent on macrophages (18). To determine if macrophages alsoregulate TLR5-induced corneal inflammation, corneas of transgenicMafia mice were abraded, flagellin was added topically, and cyto-kine production was examined after 3 h, which is prior to neutrophilrecruitment to the corneal stroma after exposure to LPS (8, 10, 26).Mafia mice express a genomic construct that comprises a mem-brane-bound suicide receptor with a cytoplasmic Fas domain un-der control of a c-fms promoter in macrophages and dendritic cellpopulations and which undergo apoptosis upon AP20817-mediateddimerization of the FK-binding protein (18, 22). As shown in Fig.1B, flagellin stimulated CXCL1 and IL-1a production in the cor-neas of untreated mice, whereas cytokine production was com-pletely ablated in AP20817-treated mice. Similar results are shownfor LPS.To examine the role of c-fms+ cells in P. aeruginosa keratitis,

corneas of untreated and AP20817-treated Mafia mice (c-fmscells) were abraded and infected with P. aeruginosa strain PAO1expressing the red fluorescent protein mCherry. After 24 h, cor-neas were examined by bright-field microscopy for corneal opacifi-cation and by fluorescent microscopy to detect eGFP+ (c-fms+) cellsand mCherry-expressing bacteria. As shown in Fig. 1C, cornealopacification was apparent within 24 h of infection, and there wasa pronounced infiltrate of c-fms+,eGFP+ cells in untreated mice, withmCherry-expressing P. aeruginosa localized to the central region ofthe cornea (site of inoculation). In marked contrast, AP20817-treatedMafia mice had less corneal opacification, eGFP+ cells were notdetected, and mCherry+ bacteria were present throughout thecornea, indicating impaired cellular infiltration and bacterial clear-ance. Quantification of CFUs showed a significantly higher bac-terial load in AP20817-treated (cfms+ cell depleted) mice com-pared with normal Mafia mice postinfection with either PAO1 orPAO1ΔfliC (Fig. 1D, 1E). Similarly, AP20817-treated Mafia miceinfected with 19660 had significantly higher CFUs than untreatedMafia mice (Fig. 1F). To examine the role of macrophages inneutrophil infiltration to the cornea during P. aeruginosa keratitis,corneas were dissected 24 h postinfection, and MPO productionwas measured. Fig. 1G shows significantly lower MPO levelsin AP20817-treated Mafia mice compared with untreated Mafiamice. We also measured cytokine production in the cornea 3 hpostinfection with 19660. As shown in Fig. 1H, P. aeruginosa-induced CXCL1 and IL-1a production was completely ablated in

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AP20817-treated Mafia mice. Taken together, these findings dem-onstrate that c-fms+ cells regulate early cytokine and chemokineproduction and subsequent neutrophil recruitment to the cornealstroma and inhibition of bacterial growth and survival.

TLR4 regulates keratitis caused by ΔfliC mutants, but notflagellate strains of P. aeruginosa

To determine the role of TLR4 in P. aeruginosa keratitis, corneasof TLR42/2 mice, TLR4Lps-d mice (which have a point mutationin TLR4), and mice deficient in the TLR4 coreceptor MD-2 (27)were abraded and infected with parent, ΔfliC 19660, or PAO1strains. Fig. 2A–D shows that CFUs recovered from mice withmutations in TLR4 or MD-2 were significantly elevated post-infection with aflagellar mutants 19660ΔfliC or PAO1ΔfliC whencompared with control C57BL/6 or BALB/c mice. In contrast,there was no significant difference in CFUs between control miceand TLR42/2, MD-22/2, or TLR4Lps-d infected with 19660 orPAO1 (expressing flagellin) (Fig. 2E–H). Consistent with elevatedCFUs in TLR42/2 mice, ΔfliC-infected corneas had less cornealopacification (Fig. 2I) and neutrophil infiltration (Fig. 2J).To ascertain whether TLR4 regulates early production of pro-

inflammatory and chemotactic cytokines, corneas of infectedC57BL/6 and TLR42/2 mice were dissected 3 h after inoculationwith 19660ΔfliC and homogenized, and cytokine concentrationwas measured by ELISA. Fig. 2K shows that CXCL1/KC and IL-

1a were elevated in infected corneas compared with traumacontrols at this time point; however, production of both cytokineswas significantly lower in infected TLR42/2 compared withC57BL/6 corneas.Because TLR4 is expressed on F4/80+ corneal macrophages

during infection, we generated bone marrow chimeras using le-thally irradiated C57BL/6 mice as recipients and donor bonemarrow cells from eGFP+TLR42/2 or eGFP+ C57BL/6 mice. Fourweeks after reconstitution, corneas were infected with 1 3 103

19660ΔfliC, and bacterial recovery was assessed after 48 h. Fig.2L shows significantly higher CFUs in C57BL/6 recipient micegiven TLR42/2 donor cells compared with mice given C57BL/6donor cells and, conversely, fewer eGFP+ cells in the corneas ofmice given TLR42/2 bone marrow cells (Fig. 2M). Together, thesefindings demonstrate that, in the absence of flagellin, TLR4 onbone marrow derived cells regulates cytokine production, cellularinfiltration, and bacterial growth in P. aeruginosa keratitis.

TLR4/52/2 mice exhibit an impaired host response to cornealinfection with P. aeruginosa that express flagella

To determine whether TLR5 activation induces an inflammatoryresponse in the cornea, we abraded the corneal surface of C57BL/6and TLR52/2 mice, stimulated the corneal surface with LPS orflagellin, and examined cellular infiltration to the corneal stromaby in vivo confocal microscopy and quantification of neutrophils

FIGURE 1. Role of macrophages in P. aeruginosa keratitis. A, Corneas of C57BL/6 mice were infected with 1 3 103 PA19660. After 18 h, corneas were

pooled from five mice and treated with collagenase, and cells were incubated with F4/80 and with Abs to TLRs and examined by flow cytometry. F4/80+

cells are shown with relative expression of TLR2, TLR4, and TLR5. In a repeat experiment, the percentage of F4/80+ cells that were TLR2+, TLR4+, and

TLR5+ cells were 73.6, 41.2, and 82.4, respectively. B–H, Mafia mice were treated with AP20817, which depletes macrophages and dendritic cells, and

corneas were either stimulated with purified LPS or flagellin or infected with P. aeruginosa 19660 or PAO1 flagellin-expressing strains or ΔfliC mutants. B,

CXCL1/KC and IL-1a from corneas 3 h after stimulation with flagellin or LPS. C, Representative corneas of AP20817 or untreated Mafia mice 24 h

postinfection with PAO1 expressing mCherry. Eyes were examined by bright-field microscopy (upper panels) and by fluorescence to visualize infiltrating

eGFP+ macrophages and dendritic cells (central panels) or mCherry expressing PAO1 (lower panels) (original magnification 320). D and E, CFUs 24 h

postinfection with PAO1 (D) or PAO1ΔfliC (E). F and G, Mice were infected with strain 19660, and CFUs (F) and MPO production (G) were measured 24 h

postinfection. H, CXCL1/KC and IL-1a in corneas 3 h postinfection with 19660 or 19660ΔfliC. Data in B and D–H are mean 6 SEM of five mice per

experiment. Similar results were found in three repeat experiments.



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in corneal sections. As shown in Fig. 3A, LPS induced a pro-nounced cellular infiltration compared with trauma (PBS) controlsin C57BL/6 and TLR52/2 corneas, which are both strains, de-tectable as small, highly refractive cells in the corneal stroma.In contrast, cellular infiltration was detected in flagellin-treatedC57BL/6 but not TLR52/2 corneas. Similarly, flagellin inducedneutrophil recruitment to the corneal stroma of C57BL/6 but notTLR52/2 mice, whereas there was no difference in neutrophilnumbers between LPS-stimulated C57BL/6 and TLR52/2 corneas(Fig. 3B). Cytokine production in the cornea also showed ligandspecificity (Fig. 3C), with significantly reduced CXCL1/KC andIL-1a in TLR52/2 corneas after stimulation with flagellin. Takentogether, these observations clearly demonstrate that flagellin in-duces TLR5-dependent corneal inflammation.To determine the role of TLR5 in P. aeruginosa keratitis, cor-

neas of C57BL/6, TLR52/2, and TLR4/52/2 mice were abradedand infected with 1 3 103 19660 (expressing flagella), and CFUswere quantified after 48 h. As shown in Fig. 3D, corneal opaci-fication was lower in TLR4/52/2 mice compared with C57BL/6and TLR52/2 mice. Consistent with this observation, TLR4/52/2

mice had impaired neutrophil infiltration to the corneal stromacompared with C57BL/6 and TLR52/2 mice (Fig. 3E). Further-more, bacterial recovery was significantly elevated in TLR4/52/2

mice compared with C57BL/6 or TLR52/2 mice (Fig. 3F), in-

dicating impaired bacterial killing in the double knockout mice.To examine the role of TLR4 and TLR5 in cytokine production,C57BL/6, TLR52/2, and TLR4/52/2 corneas were dissected 3 or18 h postinfection with PA19660, and cytokines were measured byELISA. As shown in Fig. 3G, CXCL1 and IL-1a production wereelevated in C57BL/6 and TLR52/2 corneas at 3 h postinfection,and IL-1b production was elevated in both mouse strains at 18 hpostinfection; however, production of each of these cytokines wassignificantly lower in corneas from infected TLR4/52/2 mice.Given that TLR2 is expressed on corneal macrophages (Fig. 1A),

TLR2 ligands induce an inflammatory response in the cornea (8),and TLR2 has a regulatory role in P. aeruginosa lung infections(28, 29), we also examined the role of TLR2 in P. aeruginosakeratitis. C57BL/6 and TLR22/2 corneas were infected with19660ΔfliC, and CFUs were quantified as before. There was nosignificant difference in CFU recovery between TLR22/2 andC57BL/6 mice on day 1 (Fig. 3H) or day 2 postinfection or oncellular infiltration (data not shown), indicating that there is norole for this receptor in P. aeruginosa keratitis.Taken together with results from Fig. 2, these findings indicate that

the host response in the cornea to flagella-expressing P. aeruginosacan be initiated by either TLR4 or TLR5, because the absence of bothreceptors is required to inhibit cytokine production, neutrophil in-filtration, and bacterial killing.

FIGURE 2. Role of TLR4 and MD-2 in P. aeruginosa keratitis. Corneas of C57BL/6, TLR42/2, MD-22/2, BALB/c, and TLR4Lps-d mice were abraded

and infected with either 1 3 105 PAO1 or PAO1ΔfliC or with 1 3 103 19660 or 19660ΔfliC. After 24 and 48 h, total CFUs per eye were assessed. A–D,

CFUs from mice infected with ΔfliC strains; E–H, CFUs from mice infected with parent strains. I and J, Representative C57BL/6 and TLR42/2 eyes on day

1 postinfection with 19660ΔfliC showing corneal opacification (I) and cellular infiltration (J). I, Original magnification320. J, Sections were either stained

with H&E (left panels) or immunostained with NIMP-R14 to detect neutrophils (right panels). Original magnification3400. K, Corneas were dissected and

homogenized 3 h postinfection with 1 3 103 19660ΔfliC, and CXCL1/KC and IL-1a were measured by ELISA. L and M, Bone marrow chimera mice were

generated using donor C57BL/6/eGFP+ or TLR42/2/eGFP+ bone marrow cells to reconstitute irradiated C57BL/6 recipients. Corneas were infected with

13 103 19660ΔfliC, and CFUs were examined after 48 h (L).M, Representative corneas with infiltrating eGFP+ cells. Data in A–H and K are mean6 SEM

of five corneas per group; data points in L represent individual corneas. Each experimental condition was repeated at least twice with similar results. ac,

anterior chamber; epi, corneal epithelium; Inoc, inoculum.



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P. aeruginosa activates both the TRIF- and TIRAP/MyD88-dependent pathways in bone marrow-derived macrophages

Having shown that corneal macrophages express TLR4 and TLR5and that c-fms+ macrophages, dendritic cells, and TLR4+ donor

bone marrow cells regulate P. aeruginosa growth in the cornea, we

next examined the role of TIR containing adaptor molecules in

TLR4 activation of bone marrow-derived macrophages. TLR4 is

the only member of this receptor family that uses the TRIF

pathway in addition to the MyD88 signaling pathway (30, 31) and

also requires MyD88 adaptor-like/TIRAP to recruit MyD88 to

the TIR domain of the receptor (32–34). We therefore derived

macrophages from bone marrow cells isolated from C57BL/6,

MyD882/2, TIRAP2/2, and TRIF2/2 mice, stimulated the cells

with antibiotic-killed P. aeruginosa 19660 or 19660ΔfliC or with

LPS, and measured NF-kB p65 translocation to the nucleus, IkB,

and IRF3 phosphorylation and cytokine production.Fig. 4A shows NF-kB p65 translocation in C57BL/6 macro-

phages incubated 15 min with 19660 ΔfliC, whereas p65 remained

in the cytoplasm in TIRAP2/2 and MyD882/2 macrophages and

was not detected in the nucleus until 60 min after stimulation.

Macrophages from TRIF2/2 and IL-1R12/2 mice had the same

time course of p65 translocation as C57BL/6 macrophages. Sim-

ilar results were found after LPS stimulation (data not shown).

CXCL1/KC production, which is dependent on NF-kB, was alsoinhibited in TIRAP2/2 and MyD882/2 macrophages (Fig. 4B).Conversely, CCL5/RANTES, which is dependent on the TRIFpathway (35, 36), was inhibited in TRIF2/2 but not TIRAP2/2 orMyD882/2 bone marrow macrophages (Fig. 4B).Bone marrow-derived macrophages were also processed for

Western blot analysis to detect P-IkB and IRF3. As shown inFig. 4C, left panels, P-IkB was detected in C57BL/6 macrophages15 min after stimulation with LPS, 19660, or 19660Δflic andremained elevated at 60 min. However, consistent with p65 trans-location, TIRAP2/2 and MyD882/2 macrophages stimulated withLPS or 19660ΔfliC had delayed Ikb phosphorylation comparedwith C57BL/6 macrophages (Fig. 4C). The time course of Ikbphosphoryation in TRIF2/2 and IL-1R12/2 macrophages wassimilar to C57BL/6 macrophages. To determine whether the TRIFpathway is activated, IRF3 phosphorylation was measured inC57BL/6 and TRIF2/2 macrophages. Fig. 4C, center panels, showsthat in C57BL/6 mice P-IRF3 was detected 30 or 60 min afterincubation with LPS, 19660, or 19660ΔfliC. Although there was nodifference in the time course of IRF3 phosphorylation in TIRAP2/2,MyD882/2, and IL-1R12/2 macrophages, there was no detectableP-IRF3 in TRIF2/2 macrophages. Together, these findings dem-onstrate that P. aeruginosa activates both the TIRAP/MyD88-dependent and TRIF-dependent (MyD88-independent) pathways

FIGURE 3. Role of TLR5 in corneal inflammation and P. aeruginosa keratitis. A–C, Corneas of C57BL/6 and TLR52/2 mice were abraded and treated

with LPS or flagellin. A, Representative 24-h images of the central corneal stroma captured using in vivo confocal microscopy. Infiltrating cells are highly

reflective (original magnification3200). B, Neutrophil numbers per 5-mm corneal section 24 h after exposure to LPS or flagellin. C, CXCL1/KC and IL-1a

production in corneas 3 h after exposure to LPS or flagellin. D–G, Corneas of C57BL/6, TLR52/2, and TLR4/52/2 mice were infected with 13 103 19660.

D, Representative eyes with corneal opacification 24 h postinfection (original magnification 320); E, H&E and neutrophil staining after 24 h (original

magnification 3400); F, CFUs after 24 h; and G, CXCL1/KC and IL-1a production in corneas 3 h postinfection and IL-1b production in corneas 18 h

postinfection. H, CFUs in TLR2–/– mice are infected with ΔfliC 19660. Data in B, C, and G are mean6 SEM of five corneas per group; data points in F and

H represent individual corneas. Each experiment was repeated at least twice with similar results.



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in bone marrow macrophages and indicate that they use the samepathways in bone marrow-derived macrophages in the cornea.

P. aeruginosa keratitis is regulated by TRIF and MyD88 but notTIRAP

Given that P. aeruginosa activates TRIF, MyD88, and TIRAP inbone marrow macrophages, we next examined the role of theseadaptor molecules in P. aeruginosa keratitis. To determine whetherTIRAP is required for LPS-induced corneal inflammation, corneasof C57BL/6 and TIRAP2/2mice were abraded and stimulated witheither LPS or flagellin. After 24 h, cellular infiltration to the cornealstroma was visualized by in vivo confocal microscopy, and neu-trophils in the cornea were quantified by immunohistochemistry.Fig. 5A shows a cellular infiltrate in the central corneal stroma ofLPS-treated C57BL/6 but not TIRAP2/2 corneas, whereas therewas no difference in cellular infiltration in mice treated with fla-gellin. Similarly, LPS induced neutrophil recruitment to the cornealstroma of C57BL/6 but not TIRAP2/2 mice, whereas there wasno difference in flagellin stimulated corneas (Fig. 5B), therebydemonstrating an essential role for TIRAP in LPS/TLR4 cornealinflammation.To determine if these adaptor molecules regulate P. aeruginosa-

induced cytokine production, corneas of C57BL/6, MyD882/2,

TIRAP2/2, and TRIF2/2 mice were infected with 1 3 103 19660ΔfliC, and cytokine production was measured after 3 h. As shown inFig. 5C, CXCL1 and IL-1a were elevated in P. aeruginosa-infectedC57BL/6 corneas compared with trauma controls. However, cyto-kine production was completely ablated in MyD882/2 corneas andwas significantly reduced in TIRAP2/2 and TRIF2/2 comparedwith infected C57BL/6 corneas. These findings are consistent withdata shown in Fig. 4 for bone marrow macrophages in vitro andindicate that all of these adaptor molecules are required for earlycytokine production in the cornea.To determine the role of the TIRAP/MyD88 pathway on the

outcome of P. aeruginosa keratitis, corneas of C57BL/6, MyD882/2,and TIRAP2/2 mice were infected with 1 3 103 19660 or 19660ΔfliC, and cellular infiltration, CFUs, and corneal opacification wereexamined as before. As shown in Fig. 5D, there was no difference incorneal opacification between C57BL/6 and TIRAP2/2mice after 48h, whereas MyD882/2 had less disease. Consistent with this obser-vation, there was no significant difference in CFUs between C57BL/6and TIRAP2/2 mice infected with either 19660 or 19660 ΔfliC,whereas CFUs recovered from MyD882/2 mice were significantlyelevated postinfection with either strain (Fig. 5E, 5F). Cellularinfiltration to MyD882/2 corneas was diminished compared withC57BL/6 mice (Fig. 5G), and bacteria were detected in the anterior

FIGURE 4. Role of adaptor molecules and IL-1R1 in P. aeruginosa activated macrophages. Macrophages were derived from bone marrow cells isolated

from C57BL/6, TIRAP2/2MyD882/2, TRIF2/2, and IL-1R12/2 mice and stimulated with LPS or with antibiotic killed 19660 or 19660ΔfliC. A, Rep-resentative wells showing translocation of NF-kB to the nucleus. Macrophages were incubated with killed 19660ΔfliC, and at indicated time points, cells

were fixed and immunostained with Ab to the p65 subunit of NF-kB. Data are indicative of two repeat experiments with triplicate wells. B, CXCL1/KC and

CCL5/RANTES production by bone marrow macrophages stimulated 3 h with 19660ΔfliC. C, Bone marrow macrophages were incubated with LPS, 19660,

or 19660ΔfliC. At indicated times, cells were harvested and processed for Western blot analysis using Abs to P-IkB, P-IRF3, and b-actin.



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corneal stroma ofMyD882/2mice but not controlmice. Therewas nosignificant difference in MPO production or cellular infiltration be-tween C57BL/6 and TIRAP2/2 corneas (data not shown).To examine the role of TRIF in P. aeruginosa keratitis, C57BL/6

and TRIF2/2 mice were infected with 1 3 103 19660ΔfliC. Asshown in Fig. 5H, corneal opacification was decreased in TRIF2/2

compared with C57BL/6 corneas. Similarly, MPO production wassignificantly lower in TRIF2/2 mice, and CFUs were significantlyhigher compared with C57BL/6 corneas infected with 19660 ΔfliC(Fig. 5I). In contrast, there were no differences in MPO or CFUsbetween TRIF2/2 and C57BL/6 mice infected with 19660 (Fig.5J). As CCL5/RANTES production is TRIF-dependent (Fig. 4B)(36), we also measured this cytokine in TRIF2/2 corneas at 18 hpostinfection. As shown in Fig. 5K, CCL5 was elevated in infectedC57BL/6 corneas compared with trauma controls and was sig-nificantly reduced in TRIF2/2 corneas. There was no difference inIL-1b production between infected C57BL/6 and TRIF2/2 cor-neas (Fig. 5K).In summary, these data show that TIRAP, MyD88, and TRIF

all contribute to the host response in P. aeruginosa keratitis;

however, the requirement for TIRAP was only apparent in early,macrophage-dependent cytokine production, and activation of theTRIF pathway was only revealed in the absence of flagella.

P. aeruginosa keratitis is regulated by IL-1R1, IL-1a, and IL-1b

Given that TIRAP does not appear to regulate keratitis in vivoand that TLR5 is only involved in the presence of flagella, we nextexamined whether IL-1R1 regulates P. aeruginosa keratitis as thisreceptor signals through MyD88 in the absence of TIRAP (30), andIL-1a and IL-1b are produced during corneal infection. Corneasof C57BL/6 and IL-1R12/2 mice were infected with 19660 or19660ΔfliC, and cytokine production, CFUs, corneal opacification,and cellular infiltration were examined as described above.As shown in Fig. 6A and 6B, CXCL1/KC and IL-1a production

3 h postinfection with 19660ΔfliC was significantly lower in IL-1R12/2 mice compared with C57BL/6. Conversely, CFUs weresignificantly higher in IL-1R12/2 mice compared with C57BL/6mice infected with 19660ΔfliC (Fig. 6C) or 19660 (Fig. 6D).Corneal opacification and cellular infiltration to the corneal stromawere also lower compared with C57BL/6 mice (Fig. 6E).

FIGURE 5. Role of TRIF, TIRAP, and MyD88 in P.aeruginosa keratitis. A, Representative in vivo confocal microscopy of C57BL/6 and TIRAP2/2 mice

showing light refractive cells in the central corneal stroma 24 h after stimulation with LPS or flagellin (Fla) (original magnification 3200). B, Neutrophils/

5-mm corneal sections of C57BL/6 and TIRAP2/2 mice at the same time point. C, Cytokine production in corneas 3 h postinfection with 1 3 103

19660ΔfliC. D, Representative C57BL/6, TIRAP2/2, and MyD882/2 corneas 1 day postinfection. E and F, CFUs from C57BL/6, TIRAP2/2, and

MyD882/2 mice infected with either 19660ΔfliC (E) or with 19660 (F). G, Representative histological sections of C57BL/6 and MyD882/2 corneas 24 h

postinfection with 19660 showing impaired cellular infiltration in MyD882/2 corneas. At higher magnification, bacteria can be detected in the basal

epithelium and anterior stroma. Original magnification 3400 (upper panels); 31000 (lower panels). H, Representative corneas of C57BL/6 and TRIF2/2

mice 24 h after corneal infection with 1 3 103 19660 ΔfliC. I and J, MPO and CFUs from C57BL/6 and TRIF2/2 mice infected with 19660ΔfliC (I) or

19660 (J). K, CCL5/RANTES and IL-1b production in C57BL/6 and TRIF2/2 corneas 18 h postinfection with 19660ΔfliC. D and H, Original magni-

fication 320. Data in C and K are mean 6 SEM of five corneas per group. Data points in B, E, F, I, and J represent individual corneas; experiments were

repeated three times with similar results. epi, corneal epithelium.



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As IL-1a and IL-1b are produced during P. aeruginosa kera-titis, we next infected mice deficient in IL-1 a, IL-1b, or bothcytokines with parent strain19660, and CFUs were examined. Asshown in Fig. 6F and 6G, CFUs recovered from IL-1R12/2 andIL-1a/b2/2, but not IL-1a2/2 or IL-1b2/2 mice, were signifi-cantly higher than C57BL/6 mice on days 1 and 2 postinfection.Similarly, IL-1a/b2/2 corneas showed impaired cellular infiltrationand decreased corneal opacification (Fig. 6H).Taken together, these data show that either IL-1a or IL-1b can

activate IL-1R1 in P. aeruginosa keratitis and that IL-1R1 has anessential role in regulating the inflammatory response to P. aer-uginosa in the cornea. In addition, because there is no differencein response in mice infected with 19660 or 19660ΔfliC, thesefindings also indicate that IL-1a, IL-1b, and IL-1R1 function in-dependently of flagellin/TLR5 responses.

DiscussionAlthough the normal mammalian cornea was long thought to bedevoid of bone marrow-derived cells, a number of studies haveshown that macrophages and dendritic cells are resident in this tissue,with macrophages being the predominant cell type in the cornealstroma (37–41). In the current study, we present several lines ofevidence supporting the concept that resident bone marrow-derivedcells and especially macrophages have an important role in regu-lating the outcome of corneal infection with P. aeruginosa. First,F4/80+ macrophages are the predominant cells in the corneal stromathat express surface TLR4 and TLR5 (and TLR2); second, de-

pletion of macrophages results in impaired neutrophil infiltrationand bacterial clearance; third, the absence of TLR4 on donor bonemarrow-derived cells results in impaired cellular infiltration andbacterial killing; and fourth, macrophages incubated with P. aeru-ginosa stimulate TLR4/TIRAP/MyD88- and TLR4/TRIF-dependentNF-kB translocation and cytokine production.

FIGURE 6. Role of IL-1R1, IL-1a, and IL-1b in P.

aeruginosa keratitis. Corneas of C57BL/6 and IL-

1R12/2 mice were abraded and infected with 1 3 103

19660 or 19660ΔfliC. CXCL1/KC (A) and IL-1a (B)

production 3 h postinfection with 19660ΔfliC. C and D,

CFUs 24 h postinfection with 19660ΔfliC (C) or 19660

(D). E, Representative eyes showing corneal opacifi-

cation and cellular infiltration day 1 postinfection with

19660 (original magnification 320 for corneas; 3400

for histology). F and G, CFUs from eyes of IL-1a2/2,

IL-1b2/2, and IL-1a/b2/2 mice days 1 or 2 post-

infection with 19660. G, Representative eyes on day 2

postinfection (original magnification 320). Data in A

are mean 6 SEM from five corneas per group, and data

points in CFU graphs (C, D, F, G) represent individual

eyes. These experiments were repeated three times with

similar results.

FIGURE 7. Proposed sequence of events in P. aeruginosa keratitis. See

text for details.



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Taken together, these data are consistent with the sequence ofevents shown in Fig. 7 in which P. aeruginosa LPS and flagellininitiate the host response by activating TLR4/MD-2 and TLR5 onresident macrophages in the corneal stroma. TLR5/MyD88, TLR4/MyD88 adaptor-like/TIRAP, and TLR4/TRIF signaling pathwaysall induce NF-kB translocation to the nucleus and transcription ofproinflammatory and chemotactic cytokines. These include CXCL1/KC and probably CXCL2, 5, and 8, which recruit neutrophils fromlimbal capillaries to the corneal stroma, and IL-1a and IL-1b, whichthen feedback to activate the IL-1R1/MyD88 pathway in macro-phages and most likely also in resident corneal epithelial cells andstromal fibroblasts. TLR5 on corneal epithelial cells may also con-tribute to the host response (13).Once in the corneal stroma, neutrophils actively phagocytose

and killP. aeruginosa; however, neutrophil degranulation and releaseof proteolytic enzymes and reactive oxygen species can cause ex-tensive tissue damage as part of their likely role in digesting tissue torestrict bacterial dissemination (42). In the cornea, neutrophil de-granulation induces apoptosis of resident keratocytes and cornealendothelium that regulate corneal transparency, resulting in loss ofcorneal clarity and visual impairment. The phenotype of immuno-competent mice is therefore early cytokine production, cellular re-cruitment to the corneal stroma, and control of bacterial replication.These mice exhibit early corneal opacification coincident with neu-trophil and macrophage infiltration to the corneal stroma. Althoughthese corneas develop pronounced opacification, they do not perfo-rate. In contrast, blocking the TLR or IL-1R1 response at any of thesestages results in delayed cellular infiltration (and corneal opacifica-tion), unimpaired bacterial growth, and corneal perforation.The results of our current studies are largely in agreement with

those of Huang et al. (43), who reported a role for TLR4 in reg-ulating P.aeruginosa keratitis. However, in contrast to our find-ings, these investigators reported a role for TLR4 in regulatinggrowth of the parent 19660 strain. They also showed that cytokineproduction and neutrophil infiltration were elevated rather thaninhibited (as shown in the current study) in TLR4lps-d mice andattributed impaired microbial killing to decreased expression ofantimicrobial peptides (43). The discrepancy between those studiesand data presented in this paper does not appear to be related tousing TLR4lps-dmice on a BALB/c background (43, 44), becausewefound the same role for flagellin in TLR4lps-d mice. Also consistentwith our findings, Hazlett and coworkers (45) used chlodronate-coated liposomes to deplete macrophages in the cornea and inhibi-ted bacterial killing; however, that study also reported increasedneutrophil infiltration to the cornea in the absence of macrophages,which is contrary to our observations.In addition to P. aeruginosa keratitis, our data are mostly in

agreement with P. aeruginosa-induced pulmonary disease. Thesestudies show that bacteria activate TLR4 and TLR5 on alveolarmacrophages and lung epithelial cells (28), and that TLR4 andTLR5 regulate inflammation and growth of ΔfliC bacteria in thelungs (28, 46–48). Also consistent with our data, TIRAP2/2 micehad impaired cytokine production in the lungs, but showedno difference in bacterial killing, whereas TRIF2/2 mice hadimpaired cytokine production and bacterial killing although thesestudies used flagellate P.aeruginosa (49, 50). TIRAP2/2 andTRIF2/2 mice infected with the aflagellar bacteria Klebsiellapneumonia also have impaired cytokine production and are moresusceptible to lung disease (49, 51–53).In examining the role of adaptormolecules in bonemarrow-derived

macrophages, we found that IRF3 phosphorylation was ablated inTRIF2/2 macrophages, indicating direct activation of the TLR4/TRIF pathway by P. aeruginosa. Delayed translocation of NF-kB(60 min compared with 15 min for C57BL/6 macrophages) is

consistent with the requirement for TLR4 internalization and lo-calization to endosomal compartments, where TRIF and a fourthadaptor molecule, Toll/IL-1R intracellular domain-contain adaptor-inducing IFN-b–related adaptor molecule (TRAM), are recruited(54). Conversely, early Ikb phosphorylation and p65 translocationin TRIF2/2 macrophages is likely due to TIRAP/MyD88 signaling.We therefore conclude from these and other studies (28) that P.aeruginosa activates NF-kB through the TLR4/TIRAP/MyD88 andthe TLR4/TRAM/TRIF pathways.Given the role of these adaptor molecules in vitro, we antici-

pated that all of the adaptor molecules would regulate P. aerugi-nosa keratitis. However, we found that although TRIF and MyD88had nonredundant roles, there was no effect of TIRAP deficiencyon cellular infiltration or bacterial clearance, although TIRAP2/2

mice had impaired early chemokine production. Because IL-1R12/2 mice have increased susceptibility to corneal infection withΔfliC bacteria, it appears likely that IL-1R1/MyD88 signalingoccurs in the absence of TIRAP. The apparent redundancy ofTIRAP and IL-1R1 was also detected in vitro, where there was noimpaired signaling in IL-1R12/2 macrophages. At this early timepoint (15 min after stimulation), the absence of an observed role forIL-1R1/MyD88 is likely due to TLR4/TIRAP/MyD88 signaling.Given the important role for IL-1R1 in P. aeruginosa keratitis,

we also examined the relative contribution of IL-1a and IL-1bduring infection and found that IL-1a/b2/2 mice but not singlegene knockout mice had a similar phenotype as IL-1R12/2 mice,indicating that both IL-1a and IL-1b contribute to the in-flammatory response. Hazlett and colleagues (55) showed that IL-1b is the predominant isoform in P. aeruginosa keratitis and thatcaspase-12/2 mice have decreased neutrophil infiltration (56).This is consistent with reported activation of the Ipaf inflamma-some by P. aeruginosa flagellin (57, 58).Although the current study has focused on the role of proin-

flammatory and chemotactic cytokines in regulating P. aeruginosakeratitis, it is clear that other pathways are also involved. Expressionof antimicrobial peptides, including b defensins, are elevated incorneal epithelial cells and in vivo (59, 60). Furthermore, pre-treatment of corneas with isolated flagellin inhibits P. aeruginosakeratitis by stimulating production of antimicrobial peptides, suchas cathelicidin (61, 62). In addition, expression of the cystic fibrosistransmembrane conductance regulator on corneal epithelial cells isrequired for P. aeruginosa entry into these cells and for developmentof keratitis (63). A recent study showed that P. aeruginosa infectionof mice deficient in macrophage migratory inhibition factor haveimpaired cytokine production and neutrophil infiltration to the cor-nea, although MIF–/– mice also show enhanced bacterial clearance(64). The role of T cell responses in P. aeruginosa keratitis has alsobeen extensively characterized (65). Although further studies arerequired to generate a more comprehensive understanding of howthese host responses interact to regulate bacterial replication anddevelopment of disease, our studies clearly implicate TLR/IL-1R1signaling in the early stages of corneal infection.

AcknowledgmentsWe thank Cathy Doller, Rui Zhang, Sherri Morgan, and Haowen Ge for

technical assistance and Sixto Leal for many discussions and critical review

of the manuscript.

DisclosuresThe authors have no financial conflicts of interest.

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