THE

JOURNAL • RESEARCH • www.fasebj.org

Macrophage migration inhibitory factor enhancesPseudomonas aeruginosa biofilm formation potentiallycontributing to cystic fibrosis pathogenesisAisling Tynan,* Leona Mawhinney,* Michelle E. Armstrong,* Ciaran O’Reilly,* Sarah Kennedy,†

Emma Caraher,† Karen Julicher,* David O’Dwyer,‡,§,{ Lewena Maher,‡,§,{ Kirsten Schaffer,‡,§,{

Aurelie Fabre,‡,§,{ Edward F. McKone,‡,§,{ Lin Leng,k Richard Bucala,k Jurgen Bernhagen,#,** Gordon Cooke,†,1

and Seamas C. Donnelly*,1,2

*Department of Medicine, Tallaght Hospital, Trinity College, Dublin, Ireland; †Department of Science, Centre for Microbial Host Interactions,Institute of Technology Tallaght, Dublin, Ireland; ‡St. Vincent’s University Hospital, §School of Medicine and {UCD Conway Institute ofBiomolecular and Biomedical Research, University College Dublin, Dublin, Ireland; kInternal Medicine, Yale School of Medicine, New Haven,Connecticut, USA; #Institute of Biochemistry and Molecular Cell Biology, RWTH Aachen University, Aachen, Germany; and **VascularBiology, Institute for Stroke and Dementia Research, Klinikum der Universitat Munchen, Ludwig-Maximilians-University, Munich, Germany

ABSTRACT: Macrophage migration inhibitory factor (MIF) is a key proinflammatory mediator that we have pre-viously shown to be associated with an aggressive clinical phenotype in cystic fibrosis. It possesses unique tauto-merase enzymatic activity.However, todate,nohuman-derivedsubstratehasbeenidentified thathas the capacity tointeract with this cytokine’s unique tautomerase activity. This led us to hypothesize thatMIFmayhave the capacityto interact with external substrates. We describe for the first time how Pseudomonas aeruginosa can utilize humanrecombinant MIF (rMIF) to significantly (P < 0.01) enhance its endogenous biofilm formation. Our in vivo studiesdemonstrate that utilizing a small-molecular-weight inhibitor targeting MIF’s tautomerase activity (SCD-19) sig-nificantly reduces the inflammatory response in amurine pulmonary chronic P. aeruginosamodel. In addition, weshow that in in vitro experiments, pretreatment of P. aeruginosa with rMIF is associated with reduced bacterialkilling by tobramycin. Our novel findings support the concept of an anti-MIF strategy that targets this enzymaticactivity as a potential future antibacterial therapeutic approach.—Tynan, A., Mawhinney, L., Armstrong, M. E.,O’Reilly, C., Kennedy, S., Caraher, E., Julicher, K., O’Dwyer, D., Maher, L., Schaffer, K., Fabre, A., McKone, E. F.,Leng, L., Bucala, R., Bernhagen, J., Cooke, G., Donnelly, S. C. Macrophage migration inhibitory factor enhancesPseudomonas aeruginosa biofilm formation potentially contributing to cystic fibrosis pathogenesis. FASEB J.31, 000–000 (2017). www.fasebj.org

KEY WORDS: cytokine • bacteria • respiratory infections

Macrophage migration inhibitory factor (MIF) is a keyproinflammatory cytokine that has been implicated in thepathogenesis of a variety of inflammatory diseases, in-cluding autoimmunity, asthma, cystic fibrosis (CF), andpneumonia lethality (1–5). Historically, it has also been

recognized to play a key role in sepsis, and in particulargram-negative sepsis. In seminal articles, researchers ini-tially showed that administration of specific MIFmAbs inmurine sepsis models significantly attenuated mortalitybothwhen given before and after septic insult (6, 7). It wassubsequently shown that this cytokine has the ability toaugment TLR4 cellular expression, maximize inflamma-tory cell recruitment via enhanced chemotaxis, and over-ride the glucocorticoid antiinflammatory activity (7, 8), allof which contribute either directly or indirectly to aheightened inflammatory response to injury.

In the context of Pseudomonas aeruginosa infection, theoriginal description of MIF knockout mice showed thatthese mice had less pulmonary inflammation afterP. aeruginosa infection (9). We have previously describeda functional MIF polymorphism associated with en-hancedMIF protein secretion (4). Further studies showedthat patients with genetically enhanced MIF production

ABBREVIATIONS: BAL, bronchoalveolar lavage; CF, cystic fibrosis; CFU,colony-forming unit; CLSM, confocal laser scanning microscopy; CV,crystal violet; FU, fluorescent units; GFP, green fluorescent protein; H&E,hematoxylin and eosin; ISO-1, (S,R)-3-(4-hydroxyphenyl)4.5-dihydro-5-isoxazole acetic acid methyl ester; LB, Luria-Bertani; MIF, macrophagemigration inhibitory factor; rMIF, recombinant human macrophage mi-gration inhibitory factor; SCD-19, 3-(29-methylphenyl)-isocoumarin1 These authors contributed equally to this work.2 Correspondence: Department of Clinical Medicine, School of Medicine,Trinity Biomedical Sciences Institute, Trinity College Dublin, TrinityCentre, 152-160 Pearse St., Dublin 2 4, Ireland. E-mail: seamas.donnelly@tcd.ie

doi: 10.1096/fj.201700463R

0892-6638/17/0031-0001 © FASEB 1

The FASEB Journal article fj.201700463R. Published online August 2, 2017.

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have a more aggressive clinical phenotype in CF. In thisstudy, we also found a significant association with earlyPseudomonas spp. colonization in these patients (2). Morerecently, inhibiting MIF activity has been shown to ame-liorate murine models of ocular P. aeruginosa infections(10, 11). Thus, there is an increasing body of evidenceimplicatingMIFas akeydriver of the hosts’ inflammatoryresponse after exposure togram-negative infection, and inparticular P. aeruginosa infection.

In this study, we sought to investigate whether human-derived MIF has the ability to directly affect the biologicfunctions of P. aeruginosa. In particular, we wished to ad-dress our hypothesis that P. aeruginosa has the ability tohijackspecificMIFfunctions for itsbiologicadvantage.Herewe present work revealing that this gram-negative organ-ism can utilize human MIF to enhance biofilm formation.

MATERIALS AND METHODS

Bacterial strains and culture conditions

In total 3 different strains of P. aeruginosa were used. We used 2well-characterized strains: PA01, purchased from AmericanTissue Culture Collection–LGC Standards (Middlesex, UnitedKingdom), and PA14 (a kind donation from G. O’Toole, Dart-mouth School ofMedicine,Hanover,NH,USA). For the confocalimaging, a green fluorescent protein (GFP)-taggedPA01 strain (akind donation from G. O’Toole) was used. The clinical isolate ofP. aeruginosa was obtained from K. Schaeffer (St. Vincent’s Uni-versity Hospital). Pseudomonas selective agar was (Oxoid,Basingstoke, United Kingdom) used to confirm bacterial strainsas Pseudomonas spp. All strains were routinely maintained onLuria-Bertani (LB) agar or LB broth (Sigma-Aldrich, St. Louis,MO, USA) at 37°C unless otherwise stated. Liquid cultures wereincubated overnight at 37°C on a rotary shaker at 225 rpm.

Recombinant proteins and inhibitors

For our in vitro assays, we used recombinant humanMIF (rMIF),a kind donation from R. Bucala (Yale School of Medicine, YaleUniversity, New Haven, CT, USA). We have previouslypublished ourwork on the development of an in-house novelsmall-molecular-weight inhibitor for MIF’s enzymatic ac-tivity: SCD-19 [3-(29-methylphenyl)-isocoumarin] (12). ISO-1 [(S,R)-3-(4-hydroxyphenyl)4.5-dihydro-5-isoxazole aceticacid methyl ester] is a commercially available inhibitor(VWR, Dublin, Ireland).

Biofilm formation

Biofilm formation assays were performed on the basis of themethodology from previously published work (13). In brief,starting from an overnight broth culture, a dilution containing;108 colony-formingunits (CFU)/mlwasmade. Sixteenwells ofa round-bottomed polypropylene 96-well microplate (Corning,Corning, NY, USA) were inoculated with 100 ml of this dilution.After 4 h of adhesion, the nonadhered cells were removed fromeach well and plates rinsed with PBS solution. Next, 100 ml offresh medium was added to the wells, and the plate was thenincubated for a further 24 h. The supernatants were again re-moved and the wells rinsed with PBS. Once the wells werewashed, 100ml ofPBSwasadded to eachwell, alongwith 20ml ofa commercially available resazurin solution (CellTiter-Blue;Promega, Madison, WI, USA). The plate was incubated at 37°C

for 1 h, and the fluorescence (lex 560 and lem 590 nm) wasmeasured.

Pellicle formation assay

Pellicle formation assays were performed as previously de-scribed (14). Briefly, 108 CFU/ml of PA01 was cultured in glasstest tubes with or without 100 ng/ml of rMIF in 5ml of LB brothheld stationary at 37°C. Pellicles were allowed to form at theair–liquid interface of the standing culture for 72 h. After this, themedia was carefully removed and the remaining pellicle stainedwith the addition of 1% crystal violet (CV) solution (Sigma-Aldrich) into each tube. After staining for 20min, the tubes werewashed gently with water until the unbound CV solution wasremoved from the tubes. Images of the test tubes were taken atthis time forvisual analysis.Finally, boundCVwas released fromthe pellicle by rinsing with 33% acetic acid (Sigma-Aldrich), andthe absorbance of the liquid was measured at 590 nm using aspectrophotometer.

Antibiotic treatment of preformed biofilms

After 24 h of biofilm formation, the wells were treated with50 mg/ml of tobramycin in LB broth or LB broth alone, andincubated for a further 24 h at 37°C.After incubation, thewellswerewashedwith PBS 3 times to remove any planktonic cells,and 150 ml of PBS was added to each well. The plate was thensonicated in a water bath for 10 min. The sonicated bacteriawere then serially diluted and plated on LB agar for bacterialcounts (CFU/ml).

Confocal analysis of biofilm structure

This protocol was undertaken with the help and equipment ofE.C.’s laboratory at the Institute of Technology Tallaght. Theconfocal analysis of biofilm structureswasperformedusing thebiofilm flow cell system, which has been previously describedin detail (15). Briefly, LB medium alone or conditioned with100 ng/ml MIF was pumped to the flow cell using a peristalticpump (Lennox Pump and Process; Watson Marlow, Dublin,Ireland) at a flow rate of 0.5 rpm.Overnight bacterial cultures ofGFP PA01were adjusted to an optical density of 0.1 at 600 nm bydiluting with sterile LB medium. The flow cells were inoculatedwith 300ml of culture by injection;1 cm before the flow cell. Theflowcellswere inverted to allow the bacteria to adhere to the glassslide for 1 h. The flow cells were returned upright and the clampremoved; the pumpwas started at a flow rate of 0.5 rpm and theflow cells incubated at 37°C until they were ready to be imaged.The flow cells were analyzed at 24 and 48 h using confocal laserscanning microscopy (CLSM) to take z stack images of thebiofilms. Imaris software (Bitplane, Belfast, Ireland) was usedto obtain volume per unit area (cubic micrometers per squaremicrometer); a ratio between total volume and total area coveredby biofilm was calculated. The compactness of the biofilm wasassessed as total fluorescence per volume of biofilm (16).

Chemotaxis migration assay

Primary neutrophils were isolated from 40 ml of whole bloodobtained from healthy individuals using Polymorph prep (Bio-sciences, Dublin, Ireland) as previously described (17) and di-rectly used in migration assays. Neutrophils (1 3 105) wereadded to the apical compartment of 24-well polycarbonate 5mmporous membrane Transwell inserts (Corning). One hundrednanograms per milliliter rMIF was preincubated with either100 mM of SCD-19 or ISO-1 for 30 min and then applied to the

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basal compartments of the system. The cells were incubated for16 h at 37°C, and cells that migrated to the basal side of thetranswell insert were counted.

Chronic airway infection mouse model

Specific-pathogen-free female C57BL/6 mice aged 6 to 8 wk(;20 g) were purchased from Charles River Laboratories (Wil-mington, MA, USA). They were maintained in the BiomedicalFacility, University College Dublin, under laminar airflow con-ditions. All animals had free access to standard laboratory foodand water. All housing was temperature controlled and had a12-h light–dark cycle. This work was approved by UniversityCollege Dublin animal research committee, was carried out un-der license fromtheDepartmentofHealth, Ireland, andcompliedwith the international best practice for the care and use of labo-ratory animals.

Chronic pulmonary infection with P. aeruginosawas inducedin C57BL/6 mice using a previously described methodology(5, 18). Briefly, female specific-pathogen-free mice aged 8 to10 wkwere anesthetized and inoculated intratracheally withP. aeruginosa incorporated into agar beads or sterile agarbeads. Theywere allowed to recover from anesthesia, placedinto a negative pressure isolator, and kept in standard conditionsas above. ISO-1 and SCD-19, dissolved in 5% DMSO, or controlswere administered intraperitoneally daily at a concentration of 35mg/kg throughout the course of the experiment starting on d 0.

Animals were monitored for the duration of the experiment.Total weight loss, differential cell counts, bacteria load, and his-tologic analysiswere performed as previously described (19–23).

Statistical analysis

The Student’s t test (parametric data) or Mann-Whitney test(nonparametric data) were used using GraphPad InStat 3.00(GraphPad Software, La Jolla, CA, USA). One-way analysis ofvariance with the Tukey-Kramer multiple comparisons post hoctest (parametric data) or the Kruskal-Wallis test with the Dunnmultiple comparisons posttest (nonparametric data) were usedto test for statistical significance ofdifferences betweenmore than2 experimental groups. Statistical significance was recorded atP, 0.05.

RESULTS

Investigation of biofilm formation by PA01and -14 lab strains, and clinical isolate CI58112by rMIF using resazurin assay

The reference laboratory P. aeruginosa strains PA01 andPA14 were cultured for 24 h in the presence or absence of100 ng/ml recombinant MIF (rMIF). Biofilm growth wasassessed at 24 husing the resazurin biofilmassay.Herewedemonstrate that when PA01 is grown in the presence of100 ng/ml rMIF for 24 h, there is a significant increase inbiofilm formation (195.60 6 8.83 fluorescent units(FU), P , 0.001) compared to bacteria grown in LBmedium alone (140.45 6 4.18 FU) (Fig. 1A). In addi-tion, the reference lab strain PA14 also demonstrated asignificant increase in biofilm formation when grownin the presence of 100 ng/ml of rMIF for 24 h (82.76966.74 FU, P , 0.05) compared to bacteria grown in LBmedium alone (58.82 6 2.415 FU) (Fig. 1B). A clinical

isolate CI58112 demonstrated a significant increase inbiofilm formationwhen grown in 100 ng/ml rMIF for 24 h(125.566.66 FU,P, 0.001) compared to biofilmsgrown inLB medium only (90.826 7.29 FU) (Fig. 1C).

Investigation of effect of rMIF treatment onPA01 pellicle formation using CV assay

After 72 h of culture in the presence or absence of rMIF(100 ng/ml), the pelliclewas stainedwithCV. Imagesweretaken of the stained test tubes before bound CV was re-leased (Fig. 1D). This illustrates the formation of a largerpelliclewhenPA01wasgrownin thepresenceof 100ng/mlrMIF. Subsequently, the pellicle was washed to release thebound CV, and the absorbance was measured. This dem-onstrated that therewas a significant increase in the pellicleformation of PA01 in the presence of rMIF (100 ng/ml)(0.426 0.0097, absorbance units,P, 0.01) compared to thecontrol growth (0.326 0.0043, absorbance units) (Fig. 1E).

Characterization of effects of rMIF onP. aeruginosa biofilm formation usingtime-lapse CLSM after 48-h culture

In these studies, a GFP-tagged PA01 strain was used forexperiments andbiofilmswere permitted to grow for 48 h.Images of biofilms were recorded and analyzed after 24and 48 h of culture. After 24 h of culture, representativeimages capturedbyCLSMdemonstrated thatP. aeruginosaformed a biofilm consisting of a flat biofilm interspersedwith classic mushroom-shaped biofilm structures (Fig. 2).Asignificant increase inmushroom-shapedstructureswasobserved in representative images when bacteria weregrown for 24 h in the presence of rMIF (Fig. 2B) comparedtobacteriagrown inLBcontrolmediumonly (Fig. 2A). Theimages captured by CLSM were then subjected to quan-titative analysis by Imaris software. This software appliesan algorithm to each image that uses biofilm volume,compactness, thickness, and appearance as parameters tofacilitate optimum quantification. The quantitative analy-sis demonstrated that there was a significant increase inthe volume of biofilms formed after 24 h in the presence of100 ng/ml MIF (130,553 6 44,482 mm3, P , 0.001) com-pared to control (13,3676 9334 mm3) after 24 h of growth(Fig. 2C). After 48 h, quantitative analysis was performedon images, and it was shown that there was a significantincrease in the volume of biofilms formed after 48 h in thepresence of 100 ng/ml MIF (743,239 6 191,892 mm3, P ,0.01) compared to control (103,512634,326mm3) (Fig. 2D).

Investigation of effect of rMIF-inducedbiofilm formation on P. aeruginosa survivalafter antibiotic treatment

Bacterial counts from PA01 cultures pretreated with rMIFfor 24 h and then cultured for a further 24 h in the presenceof 50 mg/ml tobramycin (6.83 106 6 3.5 3 105 CFU/ml,P , 0.01) were significantly higher than bacterial countsfromcultures thatweregrown inLBbroth onlybefore 24h

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culture in 50 mg/ml tobramycin (2.92 3 106 6 1.1 3 105

CFU/ml) (Fig. 3A).We found that that pretreatment of thereference lab strain PA14 with 100 ng/ml rMIF for 24 hbefore administration of 50 mg/ml tobramycin for an

additional 24 h significantly increased survival of bacteria(rMIF: 2.72310761.13106CFU/ml,P,0.05) comparedto bacteria grown in LB medium only before antibiotictreatment (Ctrl: 8.673 1066 1.83 106 CFU/ml) (Fig. 3B).

Figure 1. Enhanced biofilm formation by rMIF. A) Biofilms of P.aeruginosa lab strain PA01 grown for 24 h, with or without100 ng/ml rMIF, were measured by resazurin assay. Significant increase in biofilm formation was seen in biofilms formed inpresence of rMIF (100 ng/ml). Data are presented from a series of n = 5 experiments. Wilcoxon matched pairs test was used toanalyze data. ***P , 0.001. B) Biofilms of P. aeruginosa lab strain PA14 grown for 24 h, with or without 100 ng/ml rMIF, weremeasured by resazurin assay. Significant increase in biofilm formation was seen in PA14 biofilms formed in presence of rMIF(100 ng/ml). Data are presented as means 6 SEM fluorescence signals (FU) (n = 8). A paired Student’s t test was used toanalyze data. *P , 0.05. C ) Biofilms of clinical isolate CI58112 were grown for 24 h, with or without 100 ng/ml rMIF, andwere measured by resazurin assay. Significant increase in biofilm formation was seen when biofilms were formed in presenceof rMIF (100 ng/ml). Data are presented as means 6 SEM fluorescence signals (FU) (n = 8). A paired Student’s t test wasused to analyze data. ***P , 0.001 (D, E). Air–liquid interface biofilms or pellicles were allowed to form with or withoutrMIF (100 ng/ml), stained with CV, washed thoroughly, and imaged. D) Representative images are shown of CV-stainedpellicles with or without 100 ng/ml rMIF. Bound CV was released and absorbance measured at 590 nm. E) Significantincrease in biofilms grown with rMIF (100 ng/ml) was observed. Data are presented as means 6 SEM absorbance units (590 nm)(n = 8). Wilcoxon matched pairs test was used to analyze data. **P , 0.01.

Figure 2. Confocal microscopy of P. aeruginosabiofilms. GFP-tagged PA01 biofilms at 24 and48 h in absence and presence of rMIF. Usingbiofilm flow cell system, GFP-tagged PA01 wasallowed to form biofilms for 24 and 48 h at37°C in absence or presence of 100 ng/mlrMIF. biofilms were then imaged using confo-cal microscope to collect z stack compositions.Images were analyzed by Imaris software(Bitplane, Belfast, Ireland). A) Representa-tive image of biofilms grown for 24 h in LBmedium only. Scale bar, 50 mm. B) Represen-tative image of biofilms grown in presence of100 ng/ml rMIF for 24 h. Scale bars, 50 mm. C)Significant increase in volumes of biofilmsgrown for 24 h, in presence of 100 ng/mlrMIF, when calculated by Imaris software. Dataare presented as means 6 SEM cubic microme-ters (n = 10). A Mann-Whitney U test was usedto analyze data. ***P , 0.001. D) At 48 h,significant increase in volume of PA01 biofilmsgrown in presence of 100 ng/ml rMIF was demonstrated, compared to LB alone, when calculated by Imaris software. Data arepresented as means 6 SEM cubic micrometers (n = 10). A Mann-Whitney test was used to analyze data. ***P , 0.001.

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Assessment of effects of SCD-19 tautomeraseinhibitor on human polymorph nuclearneutrophil migration using chemotaxis assay

We investigated the ability of SCD-19 to decrease the mi-gration of neutrophils in response to MIF (100 ng/ml)treatment using human polymorphonuclear leukocytesfrom healthy volunteers. We demonstrated that in thepresence of rMIF, there is a significant increase in the mi-gration of neutrophils (1,927,666 6 94,686, P , 0.001)compared to the control medium (487,8756 99,573) (Fig.4). This increase is significantly reducedwhen the cells areincubatedwitheitherSCD-19 (653,167637,730,P,0.001)or ISO-1 (889,8336 20,626, P, 0.05).

Percentage weight change of mice on d7 of murine model of P. aeruginosapulmonary infection

Thepercentageweight loss of each of themice ond7of thestudy is presented in Fig. 5A. At the d 7 time point, the

uninfected control group gained weight (3.96 6 1.12%)compared to theP. aeruginosa–infected group,which lost asignificant amount of weight (21.77 6 1.01%, P , 0.01).The P. aeruginosa + DMSO group lost the most weighton d 7 of the study (22.17 6 0.92%, P , 0.01), and thegroup treated with P. aeruginosa + ISO-1 had a change inbodyweight of (21.486 1.7%,P, 0.05),with bothgroupsexperiencing significantly increasedweight loss comparedto the uninfected control group. Weight loss in the grouptreated with P. aeruginosa + SCD-19 (0.176 0.6%) was notsignificantly different from the uninfected control group.

Detection of P. aeruginosa in lunghomogenates at d 7 in murine model ofpulmonary infection

All mice were humanely killed on d 7 of the experiment.The left lungwas subsequently excised and homogenized.To detect P. aeruginosa in the lung homogenates, genomicDNA was extracted from the samples and used in a Taq-Man assay for P. aeruginosa detection. The results of theassay are shown in Table 1. P. aeruginosawas not detectedin anyof theuninfected control group samples as expected.Five of the 6 mice in the P. aeruginosa–infected group con-taineddetectable levels ofP. aeruginosa in their lungs at d 7.No samples fromeither the uninfected control groupor theSCD-19 MIF enzymatic inhibitor treatment group con-tained detectable P. aeruginosa levels in their lungs.

Differential cell counts in bronchoalveolarlavage fluid at d 7 in murine model ofP. aeruginosa pulmonary infection

The right lung was cannulated and bronchoalveolar la-vage (BAL) performed. BAL fluid was then centrifugedonto glass slides and stained using Diff-Quick. A total of

Figure 3. rMIF increases survival of (A) PA01 and (B) PA14 aftertreatment with tobramycin. Biofilms were grown for 24 h, with orwithout 100 ng/ml MIF, and then treated with 50 mg/ml oftobramycin for a further 24 h. Biofilms were sonicated, seriallydiluted, and plated on agar to analyze CFU per milliliter asmeasure of bacterial survival. A) PA01 demonstrates significantincrease in bacterial survival when it is grown in presence of MIF100 ng/ml before treatment with 50 mg/ml of tobramycin. Dataare presented as means 6 SEM CFU per milliliter (N = 10). AKruskal-Wallis with Dunn post hoc test was used for statistical analysiswhere **P, 0.01. B) Biofilms of PA14 have significantly increasedsurvival against tobramycin when pretreated with 100 ng/ml ofrMIF compared to control grown in LB alone. Data are presentedas means 6 SEM CFU per milliliter (n = 4). A Kruskal-Wallis withDunn post hoc test was used for statistical analysis where *P , 0.05.

Figure 4. SCD-19 inhibits migration of human neutrophils. Cellcounts of lower compartment of Transwell plate revealedsignificant decrease in human neutrophil migration in re-sponse to 100 ng/ml rMIF for 16 h with either 100 mM SCD-19or ISO-1. ***P, 0.001 medium with MIF only vs.medium onlyor medium with MIF and ISO-1; **P , 0.01 medium with MIFonly vs. medium with MIF and SCD-19; data presented asmeans 6 SEM cells per milliliter, n = 6, representative of at least2 experiments.

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300 cells per slide were counted to determine the differ-ential cell count. The percentage of neutrophils counted ineach sample is presented in Fig. 5B. The normal range forneutrophils in amurineBAL sample is between 5 and 10%of the total cell count. Neutrophil counts in the uninfectedcontrol group iswithin this range (6.562.1%), butboth theP. aeruginosa–infected group (22.866 1.3%,P, 0.001) andthe P. aeruginosa + DMSO group (20.36 1.26%, P, 0.01)were significantly elevated in comparison. The P. aeruginosa+ ISO-1 group showed a small decrease in neutrophils(16.6 6 2.58%) compared to the P. aeruginosa + DMSOgroup, but thiswas not statistically significant. The levelof neutrophils in the SCD-19 group was significantly de-creased (9.3 6 1.22%, P , 0.01) compared to the DMSOgroup, which were also restored to the normal range.

Histologic analysis of lungs at d 7 in murinemodel of P. aeruginosa pulmonary infection

At d 7, micewere humanely killed; the lungs of 3mice perexperimental group were perfused and fixed in 4% para-formaldehyde for histologic analysis. The tissue was sub-sequently embedded in paraffin and sliced into 5mmthicksections, which were then stained with hematoxylin andeosin (H&E) to investigate inflammatory cell infiltration.H&E histology was completed using a minimum of

6 sections from 3murine lungs.Histologic analysis of lungtissue revealed extensive inflammation, thickening of thealveolar walls, and inflammatory cell infiltration in theP. aeruginosa–infected lung (Fig. 6B) and theP. aeruginosa+DMSO lung (Fig. 6C). In contrast, the mice treated withP. aeruginosa + SCD-19 (Fig. 6E) had normal honeycombalveolar structure compared to theuninfected controlmice(Fig. 6A) and demonstrated minimal inflammatory cellinfiltration. The P. aeruginosa + ISO-1 (Fig. 6D) mice dem-onstrated an intermediate phenotype with some alveolar

Figure 5. Significant decrease in percentage weight change of mice on d 7 and presence of neutrophils after infection andtreatment with SCD-19 in murine model of PA pulmonary infection. Animals were injected 30 min before intratrachealinstillation and daily thereafter with 35 mg/kg SCD-19 (solubilized in 5% DMSO) or as indicated. Body weight was measured tomonitor extent of infection. A) Day 7 weights are expressed as percentage of start weight. PA-infected, PA + DMSO, and PA + ISO-1 groups all lost significant amount of weight compared to uninfected control group; however, SCD-19 group had not lostsignificant amount of weight at end of study. Data are presented as means 6 SEM of percentage of body weight (n = 6 per group).A 1-way ANOVA with post hoc Tukey-Kramer test was used to test for statistical differences. *P , 0.05 uninfected control vs. PA +ISO-1 **P , 0.01 uninfected control vs. PA infected or PA + DMSO. B) Differential cell counts were performed on BAL fluidfrom right lung of mice. Significant decrease in percentage of neutrophils in mice treated with SCD-19 was found in comparisonto PA-infected mice. Neutrophil numbers were assessed by differential cell count using Diff-Quick stain and are expressed asmeans6 SEM of percentage total cell count. A Kruskal-Wallis test with Dunn’s multiple comparison post hoc test was used to test forstatistical differences (n = 6 per group). ***P , 0.01, PA + DMSO vs. uninfected control; ***P , 0.01, PA + DMSO vs. PA + SCD-19. PA, P. aeruginosa.

TABLE 1. Decreased detection of P. aeruginosa in lung homogenatesof mice

Group Pseudomonas detected, n (%)

Uninfected control 0/5 (0)P. aeruginosa infected 5/6 (83)P. aeruginosa + DMSO 4/6 (67)P. aeruginosa + ISO-1 1/6 (17)P. aeruginosa + SCD-19 0/6 (0)

Decreased detection of P. aeruginosa in lung homogenates of in-fected mice treated with SCD-19. DNA was extracted from left lunghomogenate (n = 6 per group) and analyzed as per PA01 TaqMan PCRkit instructions. Results are presented as number or mice testing pos-itive for PA01. P. aeruginosa + SCD-19–treated group showed completeabsence of detectable levels of PA01 after 7 d of treatment.

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wall thickening and a minimal amount of inflammatorycell infiltration.

DISCUSSION

P. aeruginosa is a common pathogen found globally inwatery environments. Historically, it has shown a highlyeffectiveability to adapt to changinghostile environments,ultimately for its enhanced survival. It is a major cause ofhospital-acquired infections, particularly in the lungs, andchronic infection with mucoid strains in CF is associatedwith accelerated decline in lung function and increasedmorbidity and mortality (24). One of the most character-ized mechanisms by which this pathogen evades hostdefenses is via enhanced biofilm formation (25).

We here present evidence for the ability of this bacteriato hijack a human cytokine, namely MIF, for its biologicadvantage. MIF possesses unique tautomerase enzymaticactivity, and todate, nohuman-derived substratehas beenidentified that has the capacity to interact with this cyto-kine’s unique tautomerase activity. We thus decided toinvestigate whether MIF, the tautomerase enzyme, mayhave the capacity to interact with external bacterial sub-strates. This led to our novel finding that MIF could en-hance biofilm formation of P. aeruginosa by means of thisenzymatic activity and that inhibitors of this enzymaticactivitymay potentially act as an effective adjunct therapy

to antibiotics to treat human P. aeruginosa infections in thefuture.

A unique functional characteristic of MIF is that itpossesses tautomerase enzymatic activity and was origi-nally described as having the ability to tautomerasethe nonphysiologic substrates D-dopachrome and L-dopachromemethyl ester into their indolederivatives (26).ISO-1 represents the most characterized commerciallyavailable inhibitor of MIF’s tautomerase activity (27). Inthe context of gram-negative sepsis, ISO-1 has been shownto attenuatemousemortality in a classic LPSmurine sepsismodel (28). To date, no definitive physiologic substrate forMIF’s enzymatic activity has been identified in humans.This led us to hypothesize that human-derived MIF mayinteract with external bacterial derived substrates. Insupport of this, it is well recognized that enzymaticMIFhas structural homology with bacterial tautomerase en-zymes, including 4-oxalocrotonate tautomerase and 5-carboxymethyl-2-hydroxymuconate isomerase (29, 30).The original seminal work describing the phenotype ofMIF knockout mice showed that mice lacking global MIFactivity had less pulmonary inflammation in a murinemodelofpulmonary infectionwithP. aeruginosa (9). Inouroriginalworkshowing that theMIFCATTpolymorphismwas associated with more aggressive disease in CF, wealso found a significant associationwith earlier pulmonaryP. aeruginosa colonization in these patients (2). More

Figure 6. Staining of lung sections. H&Estaining was performed on murine lung sec-tions at d 7 after infection from the followinggroups: uninfected control (A), PA infected(B), PA + DMSO (C), PA + ISO-1, (D) and PA +SCD-19 (E). PA-infected mice treated withSCD-19 tautomerase inhibitor (E) displayedreduced level of inflammation compared toDMSO control mice (C). Sections from ISO-1-treated mice (D) also demonstrated decrease ininflammation compared to DMSO-treated mice(C). Images are representative of minimum of6 sections from 3 murine lungs. Scale bars,100 mm. PA, P.s aeruginosa.

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recently we published work that utilized a tautomerase-null MIF gene knock-in mouse in a chronic pulmonarymurine Pseudomonas infection model and found that thesemice had significantly less infection, bacteria load, andpulmonary injury (5). Investigatorshavepreviously shownthat blocking MIF activity in a murine ocular Pseudomonasinfectionmodel significantly reduced bacterial burden andocular inflammation (11). Further work has demonstratedthat mice deficient for CD74, the putative MIF receptor,developed milder P. aeruginosa–induced disease. Addi-tionally, topical inhibition of MIF in this model furtherpromoted recovery from disease (10). This suggests anadditional CD74-independant mechanism for the role ofMIF inPseudomonas infection.Thus, there exists supportingevidence linking MIF, its enzymatic activity, and infectionwith P. aeruginosa.

This led us to hypothesize that P. aeruginosa has theability to utilize host-derived MIF, and specifically itstautomerase enzymatic activity, for survival advantage.Our results showed that P. aeruginosa biofilm formationcould be enhancedwith the addition of rMIF and that thisenhancement could be demonstrated in multiple ways.We noted that the rMIF-enhanced biofilm formation inour studies did not occur in a concentration-dependentmanner but was consistently observed when rMIF con-centrations .50 ng/ml were used (data not shown).Concentrations of MIF in the lung vary greatly, with pre-vious studies having shown that in plasma and BAL fluidfrom septic lungs, concentrations of between 60 and100 ng/ml have been detected (31, 32). The finding thatrMIF can enhance biofilm formation raises the possibilitythat P. aeruginosa has the ability to sense the concentrationofMIF as amarker of the local inflammatory environmentand can accelerate endogenous biofilm formation whenthe local inflammatory defenses reach a certain threshold.Although this is an intriguing possibility,much additionalwork will be required to validate this proposition.

To investigate whether this novel observation of en-hanced biofilm formation confers a survival advantage tothe bacteria, we pretreated P. aeruginosa with rMIF andassessedbacterial killing after exposure to tobramycin.Wefound pretreated bacteria to have enhanced survival afterantibiotic exposure in our in vitro system. From a clinicalperspective, this raises the tantalizing possibility that tar-geting MIF’s tautomerase enzymatic activity representsa valid therapeutic strategy against this gram-negativeorganism.

We have recently developed a specific in-house set ofinhibitors for this enzymatic activity, and we have shownthat our lead compound, SCD-19, has significant antican-cer activity (20). To address the role of this unique enzy-matic activity in P. aeruginosa function, and specificallyMIF-inducedbiofilm formation,weused an animalmodelof chronic P. aeruginosa infection in which we used ourown lead inhibitor ofMIF enzymatic activity and a knowninhibitor, ISO-1.We found that SCD-19 could significantlyinhibit MIF-induced primary human neutrophil chemo-taxis both in vitro and in vivo. This is important becauseneutrophils are a source ofMIF and also have been shownto contribute to biofilm formation in the lung (33).We alsofound that SCD-19 treatment in our murine pulmonary

P. aeruginosa infection model reduces pulmonary in-flammation and bacterial burden in treated mice. Thesedata support the idea of the importance of this tautomeraseactivity in accelerating MIF-induced biofilm formation.

Enhanced MIF activity has been shown to be a keydriver of an injurious inflammatory response, and pre-viously published work, including our own, has impli-cated this cytokine in the pathogenesis of a variety ofchronic inflammatory diseases (4, 10, 11). In the context ofgram-negative sepsis, it has been shown to contribute to asustained inflammatory response by directly promotingproinflammatory cytokine release in infected organs andindirectly via its ability to first augment cellular TLR4 ex-pression, a key pattern recognition receptor for LPS (8);second to down-regulate p53, leading to a longer life spanfor these activated inflammatory cells; and third to po-tentially override the body’s own antiinflammatory glu-cocorticoid activity (31). This thus leads to a sustainedantibacterial inflammatory response. In response, P. aeru-ginosa hijacks a human proinflammatory cytokine, whichresults in enhanced survival of this gram-negative organ-ism and which confers a significant biologic advantage tothese bacteria, compared to other organisms, in this localinflammatory firestorm.

MIF has been shown to promote leukocyte recruitmentduring inflammation,which can be partly explained by itschemokine properties (32). In in vivo arthritis models, di-rect injection ofMIF induces a significant neutrophil influxinto affected joints (34). Conversely, thesemodels, on aMIFknockoutmurinebackground, showasignificant reductionof neutrophils within inflamed joints (35). More recently, aseries of elegant experiments definitively showedMIF to bea potent neutrophil chemokine in murine inflammatoryarthritis (36). Here we present data showing that SCD-19significantly inhibits neutrophil chemotaxis both in in vitrosystems utilizing primary human neutrophils and in our invivo murine model of pulmonary P. aeruginosa infection.The search for novel methods to deal with antibiotic re-sistance represents a global imperative and a significantchallenge. This work supports the targeting of MIF’s tau-tomerase activity as a valid adjunctive therapy in combat-ing bacterial infections.

ACKNOWLEDGMENTS

S.C.D. was supported by grants from Science FoundationIreland (SFI), the Health Research Board (HRB), and the IrishLung Foundation (ILF). The authors thank G. A. O’Toole(Dartmouth School of Medicine, Hanover, NH, USA) for hiskind donation of PA14- and GFP-tagged PA01. G.C. and S.C.D.share senior authorship. The authors declare no conflicts ofinterest.

AUTHOR CONTRIBUTIONS

A. Tynan, M. E. Armstrong, G. Cooke, and S. C. Donnellydesigned research; A. Tynan, L. Mawhinney, M. E.Armstrong, C. O’Reilly, K. Julicher, L. Maher, andG. Cooke performed research; S. Kennedy, E. Caraher,K. Schaffer, A. Fabre, E. F.McKone,L. Leng,R.Bucala, andJ. Bernhagen contributed new reagents or analytical tools;

8 Vol. 31 November 2017 TYNAN ET AL.The FASEB Journal x www.fasebj.org Vol.,  No. , pp:, August, 2017The FASEB Journal. 193.1.120.28 to IP www.fasebj.orgDownloaded from

A. Tynan, L. Mawhinney, C. O’Reilly, K. Julicher,D. O’Dwyer, A. Fabre, G. Cooke, and S. C. Donnellyanalyzed data; and A. Tynan, G. Cooke, and S. C. Donnellycontributed to writing the article.

REFERENCES

1. Rossi, A. G., Haslett, C., Hirani, N., Greening, A. P., Rahman, I., Metz,C. N., Bucala, R., and Donnelly, S. C. (1998) Human circulatingeosinophils secrete macrophage migration inhibitory factor (MIF).Potential role in asthma. J. Clin. Invest. 101, 2869–2874

2. Plant, B. J., Gallagher, C. G., Bucala, R., Baugh, J. A., Chappell, S.,Morgan, L., O’Connor, C.M.,Morgan, K., andDonnelly, S. C. (2005)Cystic fibrosis, disease severity, and a macrophage migrationinhibitory factor polymorphism. Am. J. Respir. Crit. Care Med. 172,1412–1415

3. Donnelly, S. C., Haslett, C., Reid, P. T., Grant, I. S., Wallace, W. A.,Metz, C. N., Bruce, L. J., and Bucala, R. (1997) Regulatory role formacrophage migration inhibitory factor in acute respiratory distresssyndrome. Nat. Med. 3, 320–323

4. Baugh, J. A., Chitnis, S., Donnelly, S. C., Monteiro, J., Lin, X., Plant,B. J., Wolfe, F., Gregersen, P. K., and Bucala, R. (2002) A functionalpromoter polymorphism in the macrophage migration inhibitoryfactor (MIF) gene associated with disease severity in rheumatoidarthritis. Genes Immun. 3, 170–176

5. Adamali, H., Armstrong, M. E., McLaughlin, A. M., Cooke, G.,McKone, E., Costello, C. M., Gallagher, C. G., Leng, L., Baugh, J. A.,Fingerle-Rowson,G.,Bucala,R. J.,McLoughlin,P., andDonnelly,S.C.(2012) Macrophage migration inhibitory factor enzymatic activity,lung inflammation, andcysticfibrosis.Am. J. Respir.Crit. CareMed.186,162–169

6. Calandra, T., Echtenacher, B., Roy, D. L., Pugin, J., Metz, C. N.,Hultner, L., Heumann, D.,Mannel, D., Bucala, R., andGlauser, M. P.(2000)Protection fromseptic shock by neutralization ofmacrophagemigration inhibitory factor. Nat. Med. 6, 164–170

7. Calandra, T., Bernhagen, J., Metz, C. N., Spiegel, L. A., Bacher, M.,Donnelly, T., Cerami, A., and Bucala, R. (1995) MIF as aglucocorticoid-induced modulator of cytokine production. Nature377, 68–71

8. Roger, T., David, J., Glauser, M. P., and Calandra, T. (2001) MIFregulates innate immune responses through modulation of Toll-likereceptor 4. Nature 414, 920–924

9. Bozza, M., Satoskar, A. R., Lin, G., Lu, B., Humbles, A. A., Gerard, C.,and David, J. R. (1999) Targeted disruption of migration inhibitoryfactor gene reveals its critical role in sepsis. J. Exp. Med. 189, 341–346

10. Zaidi, T., Reidy, T., D’Ortona, S., Fichorova, R., Pier, G., andGadjeva,M.(2011)CD74deficiencyamelioratesPseudomonasaeruginosa–inducedocular infection. Sci. Rep. 1, 58

11. Gadjeva, M., Nagashima, J., Zaidi, T., Mitchell, R. A., and Pier, G. B.(2010) Inhibition of macrophage migration inhibitory factorameliorates ocular Pseudomonas aeruginosa–induced keratitis. PLoSPathog. 6, e1000826

12. Mawhinney, L., Armstrong, M. E., O’ Reilly, C., Bucala, R., Leng, L.,Fingerle-Rowson, G., Fayne, D., Keane, M. P., Tynan, A., Maher, L.,Cooke, G., Lloyd, D., Conroy, H., and Donnelly, S. C. (2015)Macrophagemigration inhibitory factor (MIF) enzymatic activity andlung cancer.Mol. Med. 20, 729–735

13. Peeters, E.,Nelis,H. J., andCoenye,T. (2008)Comparisonofmultiplemethods for quantification of microbial biofilms grown in microtiterplates. J. Microbiol. Methods 72, 157–165

14. Merritt, J. H., Kadouri, D. E., andO’Toole, G. A. (2005)Growing andanalyzing static biofilms. Curr. Protoc. Microbiol. Chapter 1, Unit 1B.1.

15. Weiss Nielsen,M., Sternberg, C., Molin, S., and Regenberg, B. (2011)PseudomonasaeruginosaandSaccharomyces cerevisiaebiofilm inflowcells.J. Vis. Exp. (47), 2383

16. Ghafoor, A., Hay, I. D., and Rehm, B. H. (2011) Role ofexopolysaccharides in Pseudomonas aeruginosa biofilm formation andarchitecture. Appl. Environ. Microbiol. 77, 5238–5246

17. Zhou, L., Somasundaram, R., Nederhof, R. F., Dijkstra, G., Faber,K. N., Peppelenbosch, M. P., and Fuhler, G. M. (2012) Impact ofhuman granulocyte and monocyte isolation procedures onfunctional studies. Clin. Vaccine Immunol. 19, 1065–1074

18. Hopkins, N., Cadogan, E., Giles, S., and McLoughlin, P. (2001)Chronic airway infection leads to angiogenesis in the pulmonarycirculation. J. Appl. Physiol. 91, 919–928

19. Reber, L. L., Daubeuf, F., Plantinga, M., De Cauwer, L., Gerlo, S.,Waelput, W., Van Calenbergh, S., Tavernier, J., Haegeman, G.,Lambrecht, B. N., Frossard, N., and De Bosscher, K. (2012) Adissociated glucocorticoid receptor modulator reduces airwayhyperresponsiveness and inflammation in a mouse model ofasthma. J. Immunol. 188, 3478–3487

20. Kruger, T. F., Ackerman, S. B., Simmons, K. F., Swanson, R. J., Brugo,S. S., and Acosta, A. A. (1987) A quick, reliable staining technique forhuman sperm morphology. Arch. Androl. 18, 275–277

21. Leigh, R., Ellis, R., Wattie, J., Southam, D. S., De Hoogh,M., Gauldie,J., O’Byrne, P. M., and Inman, M. D. (2002) Dysfunction andremodeling of the mouse airway persist after resolution of acuteallergen-induced airway inflammation. Am. J. Respir. Cell Mol. Biol. 27,526–535

22. Nadkarni, M. A., Martin, F. E., Jacques, N. A., and Hunter, N. (2002)Determination of bacterial load by real-timePCRusing a broad-range(universal) probe and primers set.Microbiology 148, 257–266

23. Deschaght, P., De Baere, T., Van Simaey, L., Van Daele, S., De Baets,F., DeVos, D., Pirnay, J. P., andVaneechoutte,M. (2009)Comparisonof the sensitivityof culture, PCRandquantitative real-timePCRfor thedetection of Pseudomonas aeruginosa in sputum of cystic fibrosis pa-tients. BMC Microbiol. 9, 244

24. Lobo, J., Rojas-Balcazar, J. M., and Noone, P. G. (2012) Recentadvances in cystic fibrosis. Clin. Chest Med. 33, 307–328

25. Moreau-Marquis, S., Stanton, B. A., and O’Toole, G. A. (2008)Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway.Pulm. Pharmacol. Ther. 21, 595–599

26. Rosengren, E., Bucala, R., Aman, P., Jacobsson, L., Odh, G., Metz,C. N., and Rorsman, H. (1996) The immunoregulatory mediatormacrophage migration inhibitory factor (MIF) catalyzes atautomerization reaction.Mol. Med. 2, 143–149

27. Lubetsky, J.B.,Dios,A.,Han,J.,Aljabari,B.,Ruzsicska,B.,Mitchell,R.,Lolis,E., and Al-Abed, Y. (2002) The tautomerase active site of macrophagemigration inhibitory factor is a potential target for discovery of novel anti-inflammatory agents. J. Biol. Chem. 277, 24976–24982

28. Al-Abed, Y., Dabideen, D., Aljabari, B., Valster, A., Messmer, D.,Ochani, M., Tanovic, M., Ochani, K., Bacher, M., Nicoletti, F., Metz,C., Pavlov, V. A.,Miller, E. J., andTracey, K. J. (2005) ISO-1 binding tothe tautomeraseactive siteofMIF inhibits its pro-inflammatory activityand increases survival in severe sepsis. J. Biol. Chem. 280, 36541–36544

29. Bendrat, K., Al-Abed, Y., Callaway, D. J., Peng, T., Calandra, T., Metz,C.N., andBucala,R. (1997)Biochemical andmutational investigationsof the enzymatic activity of macrophage migration inhibitory factor.Biochemistry 36, 15356–15362

30. Sugimoto, H., Suzuki, M., Nakagawa, A., Tanaka, I., and Nishihira, J.(1996) Crystal structure of macrophage migration inhibitory factorfrom human lymphocyte at 2.1 A resolution. FEBS Lett. 389, 145–148

31. Song, X., Guo,M.,Wang,T.,Wang,W., Cao, Y., andZhang,N. (2014)Geniposide inhibited lipopolysaccharide-induced apoptosis by mod-ulating TLR4 and apoptosis-related factors in mouse mammaryglands. Life Sci. 119, 9–17

32. Bernhagen, J., Krohn, R., Lue, H., Gregory, J. L., Zernecke, A.,Koenen,R.R.,Dewor,M.,Georgiev, I., Schober,A., Leng,L.,Kooistra,T., Fingerle-Rowson, G., Ghezzi, P., Kleemann, R., McColl, S. R.,Bucala, R., Hickey, M. J., and Weber, C. (2007) MIF is a noncognateligand of CXC chemokine receptors in inflammatory andatherogenic cell recruitment. Nat. Med. 13, 587–596

33. Parks,Q.M., Young,R.L., Poch,K.R.,Malcolm,K.C., Vasil,M.L., andNick, J. A. (2009)Neutrophil enhancement of Pseudomonas aeruginosabiofilm development: human F-actin and DNA as targets for therapy.J. Med. Microbiol. 58, 492–502

34. Gregory, J. L., Morand, E. F., McKeown, S. J., Ralph, J. A., Hall, P.,Yang, Y. H., McColl, S. R., and Hickey, M. J. (2006) Macrophagemigration inhibitory factor induces macrophage recruitment via CCchemokine ligand 2. J. Immunol. 177, 8072–8079

35. Gregory, J. L., Leech,M. T., David, J. R., Yang, Y.H., Dacumos, A., andHickey, M. J. (2004) Reduced leukocyte–endothelial cell interactionsin the inflamedmicrocirculationofmacrophagemigration inhibitoryfactor–deficient mice. Arthritis Rheum. 50, 3023–3034

36. Santos, L. L., Fan, H., Hall, P., Ngo, D., Mackay, C. R.,Fingerle-Rowson, G., Bucala, R., Hickey, M. J., and Morand, E. F.(2011)Macrophage migration inhibitory factor regulates neutrophilchemotactic responses in inflammatory arthritis in mice. ArthritisRheum. 63, 960–970

Received for publication May 19, 2017.Accepted for publication July 17, 2017.

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10.1096/fj.201700463RAccess the most recent version at doi: published online August 2, 2017FASEB J 

  Aisling Tynan, Leona Mawhinney, Michelle E. Armstrong, et al.   fibrosis pathogenesis

biofilm formation potentially contributing to cysticaeruginosaPseudomonasMacrophage migration inhibitory factor enhances

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