Role of Excessive Inﬂammatory Response to Stenotrophomonas ... · has the potential to contribute...

INFECTION AND IMMUNITY, June 2010, p. 2466–2476 Vol. 78, No. 60019-9567/10/$12.00 doi:10.1128/IAI.01391-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Role of Excessive Inflammatory Response toStenotrophomonas maltophilia Lung Infection

in DBA/2 Mice and Implicationsfor Cystic Fibrosis�

Giovanni Di Bonaventura,1,2†* Arianna Pompilio,1,2† Roberta Zappacosta,3 Francesca Petrucci,1Ersilia Fiscarelli,4 Cosmo Rossi,2 and Raffaele Piccolomini1,2

Department of Biomedical Sciences1 and Department of Oncology and Neurosciences,3 G. d’Annunzio University ofChieti-Pescara, Chieti, Italy; Center of Excellence on Aging, G. d’Annunzio University Foundation, Chieti,

Italy2; and Cystic Fibrosis Microbiology, Pediatric Hospital Bambino Gesu, Rome, Italy4

Received 14 December 2009/Returned for modification 13 January 2010/Accepted 12 March 2010

Stenotrophomonas maltophilia is a pathogen that causes infections mainly in immunocompromised patients.Despite increased S. maltophilia isolation from respiratory specimens of patients with cystic fibrosis (CF), thereal contribution of the microorganism to CF pathogenesis still needs to be clarified. The aim of the presentstudy was to evaluate the pathogenic role of S. maltophilia in CF patients by using a model of acute respiratoryinfection in DBA/2 mice following a single exposure to aerosolized bacteria. The pulmonary bacterial load wasstable until day 3 and then decreased significantly from day 3 through day 14, when the bacterial load becameundetectable in all infected mice. Infection disseminated in most mice, although at a very low level. Severeeffects (swollen lungs, large atelectasis, pleural adhesion, and hemorrhages) of lung pathology were observedon days 3, 7, and 14. The clearance of S. maltophilia observed in DBA/2 mouse lungs was clearly associated withan early and intense bronchial and alveolar inflammatory response, which is mediated primarily by neutro-phils. Significantly higher levels of interleukin-1� (IL-1�), IL-6, IL-12, gamma interferon (IFN-�), tumornecrosis factor alpha (TNF-�), GRO�/KC, MCP-1/JE, MCP-5, macrophage inflammatory protein 1� (MIP-1�), MIP-2, and TARC were observed in infected mice on day 1 with respect to controls. Excessive pulmonaryinfection and inflammation caused systemic effects, manifested by weight loss, and finally caused a highmortality rate. Taken together, our results show that S. maltophilia is not just a bystander in CF patients buthas the potential to contribute to the inflammatory process that compromises respiratory function.

Cystic fibrosis (CF) has a peculiar set of bacterial pathogens,including Staphylococcus aureus, usually acquired in the earlystages, and Pseudomonas aeruginosa, which will eventually in-fect up to 80% of adults with CF (21). Aggressive, early treat-ment with anti-pseudomonad antibiotics results in an improve-ment in lung function and prognosis; however, the selectivepressure of antibiotics on bacterial species and the longer sur-vival of patients have been associated with the emergence ofnew pathogens, such as Stenotrophomonas maltophilia (2, 14,15, 50).

An increasing incidence of S. maltophilia isolates has beenreported in some CF centers during the last decade (2, 9, 15,22, 30). In the United States, the frequency of S. maltophiliaisolation from CF patients increased rapidly, from around 3%in 1993 to 12.1% in 2006 (21). A prevalence of up to 25% hasbeen reported in Europe (47), with rates ranging from 7 to11% in different Italian centers (34, 46). Spicuzza et al. (46), ina retrospective evaluation of a cohort of Italian CF patientsfrom 1996 to 2006, found that S. maltophilia was the only truly

emerging pathogen, as it had never been isolated until 2004,when an incidence of 7% among all patients was recorded andremained constant through 2006. In non-CF patients (e.g.,immunocompromised or intensive care unit patients), expo-sure to wide-spectrum antimicrobial drugs, long-term antimi-crobial therapy, previous pulmonary infections, and chronicrespiratory disease contribute to S. maltophilia acquisition andincrease the risk for respiratory infection with this microorgan-ism (49, 51, 52). All of these risk factors are present in the CFpopulation (49).

Despite increased S. maltophilia isolation from CF patients,its potential for pathogenicity remains undetermined becauseof conflicting clinical results from studies investigating the cor-relation between the presence of this microorganism and lungdamage. Although Karpati et al. (30) reported that prolongedinfection with S. maltophilia was generally associated withworse lung function in CF patients, most studies reported onlya mild effect on lung function (2, 14, 15, 29, 46, 49).

For these reasons, studies using in vivo models which moreclosely mimic CF pulmonary tissues are certainly needed tobetter clarify the pathogenic role of S. maltophilia in CF pa-tients.

Therefore, in the present work, we infected DBA/2 mouselungs with an S. maltophilia CF strain by using an aerosoldelivery technology in order to simulate more closely the air-borne transmission thought to be important in naturally occur-

* Corresponding author. Mailing address: Laboratory of Clinical Mi-crobiology, Center of Excellence on Aging (Ce.S.I.), G. D’Annunzio Uni-versity Foundation, Via Colle dell’Ara, 66100 Chieti, Italy. Phone: 39 0871541509. Fax: 39 0871 541520. E-mail: [email protected].

† G.D.B. and A.P. contributed equally to the experiments and thepreparation of the manuscript.

� Published ahead of print on 22 March 2010.

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ring CF pulmonary infections (53). Using this approach, forthe first time we developed a model of acute respiratory infec-tion by S. maltophilia to investigate bacterial clearance, histo-logical damage, and inflammatory response in the lungs ofinfected mice relative to those in normal controls.

MATERIALS AND METHODS

Mice. Seven-week-old female (n � 46) and male (n � 46) specific-pathogen-free DBA/2 inbred mice (Charles River Laboratories Italia, Calco, Italy) wereused in all experiments. Animals were housed, bred, and maintained in a barrierfacility unit under specific-pathogen-free conditions with a 12-h light-dark cycle.Six to 12 animals were kept in polycarbonate sterile microisolator cages andmaintained in a ventilated HEPA-filtered rack. All mice had access ad libitum tosterile acidified water and irradiated diet. All procedures involving mice werereviewed and approved by the Animal Care and Use Committee of G.d’Annunzio University of Chieti-Pescara.

Bacterial strain and growth conditions. S. maltophilia strain OBGTC9, orig-inally isolated from the sputum of a CF patient admitted to the Cystic FibrosisUnit of the Pediatric Hospital Bambino Gesu of Rome, was used in all experi-ments. This strain was selected for its strong ability to adhere to CF-derivedbronchial epithelial IB3-1 cells, a feature highly conserved in S. maltophilia CFstrains (17). Bacterial stocks were maintained at �80°C until use.

To prepare the infectious dose for nebulization, S. maltophilia was grown withagitation (130 rpm) in 200 ml of Trypticase soy broth (Oxoid SpA, GarbagnateMilanese, Milan, Italy) for 16 h at 37°C and harvested by centrifugation at3,220 � g for 10 min at 4°C. Pellets were then washed twice and resuspended incold 1% phosphate-buffered saline (PBS). Cell density was adjusted to about1.0 � 1010 to 3.0 � 1010 CFU/ml, and 8 ml of this standardized suspension wasused for nebulization. The inoculum concentration was confirmed by serialplating after each experiment.

Experimental model of infection. For airborne lung infection, a home-madeaerosol dispersal system housed in a laminar-flow biological safety cabinet wasused (Fig. 1). This device allows for infection of up to four mice simultaneously.

The standardized bacterial suspension (1.0 � 1010 to 3.0 � 1010 CFU/ml) wasnebulized (minimum flow rate, 5 liters/min; operating pressure, 60 kPa) andtransferred by a piston compressor (Hospyneb Professional; 3A Health Care) toeach of the four inhalation chambers, each consisting of a 30-ml syringe(Terumo). Before exposure to infected aerosol, mice were weighed and theninserted into the syringe, introducing the head initially and then the rest of thebody by pushing them gently and successively repositioning the plunger. Thechamber was then connected to the device for nebulization.

A standard aerosol exposure cycle consisted of 60 min for nebulization and 5min for cloud decay, and finally, mice were externally decontaminated by expo-sure for 5 min to UV irradiation.

This inhalation exposure system allowed us to deliver nebulized bacteria indistal airways uniformly throughout the lungs, as confirmed by histological anal-ysis (data not shown). Nebulization of 8 ml of a suspension containing 1.0 � 1010

to 3.0 � 1010 CFU/ml delivered a reproducible number of bacteria in both lungsand among all animals simultaneously exposed, as confirmed by culture analysis,yielding low mouse-to-mouse variability in the initial doses delivered into thelung (mean � standard deviation [SD], 4.8 � 106 � 8.6 � 105 CFU/lung;coefficient of variation, 17.8% [results are from two independent experimentswith eight animals each]).

Experimental design. The experimental layout is shown in Fig. 2. Age- andgender-matched DBA/2 mouse groups were randomized at the time of exposureto receive an aerosol with PBS containing S. maltophilia OBGTC9 (12 mice/group) or one with PBS only (6 mice/group). The general health of the mice wasmonitored daily, with mice considered unwell when a �10% weight loss orgeneral ill appearance (ruffled coat, huddled position, or lack of retreat inhandler’s presence) was observed with respect to controls. Mice were sacrificedby CO2 inhalation at 1, 2, 3, 7, and 14 days postexposure. To determine the initialbacterial deposition in the lungs, mice (n � 8) were sacrificed immediately (1 h)after the standard exposure cycle. At each time point for each group, the lungswere observed in situ for macroscopic analysis and then removed en bloc from thechest via sterile excision. In each group, 8 (infected group) or 4 (control group)lungs were randomly assigned to one of the three following outcome measures:(i) histological analysis, (ii) quantitative bacteriology, and (iii) cytokine/chemo-kine measurements. To determine whether there had been dissemination ofbacteria from the lung, resulting in bacteremia, at each time point the spleen ofeach mouse was homogenized and cultured for quantitative analysis.

Lung macroscopic and histopathological analyses. The lungs were scored bothin situ and after removal from the thoracic cavity according to the methodproposed by Johansen et al. (27), as follows: �1, normal; �2, swollen lungs,hyperemia, and small atelectasis; �3, pleural adhesion, atelectasis, and multiplesmall abscesses; and �4, large abscesses, large atelectasis, and hemorrhages.

For histopathological examination, the isolated lungs were immediately fixedin 10% neutral buffered formalin, and each was then sectioned once along thelong axis of the lobe. Tissue sections (thickness, 3 �m) were obtained from dorsallung surfaces, throughout the tissue block and at regular intervals of 50 �m.Sections were then stained with Giemsa stain, and the degree of inflammationwas scored by using a five-point system proposed by Dubin and Kolls (19). Tenfields were evaluated per lung, at low (�10), medium (�20), and high (�63 and�100) magnifications. Histopathological evaluation was done by investigatorswho were blinded to the sample origin.

Quantitative bacteriology. Pulmonary clearance and occurrence of dissemi-nated infection were monitored by quantitative bacteriology of lung and spleenhomogenates, respectively. Briefly, lungs were homogenized (24,000 � g/min) onice in 2 ml of sterile PBS by use of an Ultra-Turrax T25-Basic homogenizer(IKA-Werke GmbH & Co. KG, Germany). The homogenizer was disinfectedbetween each sample, using absolute ethanol (to avoid cross-contamination), andwas rinsed twice with sterile PBS (to avoid inhibition of bacterial growth in thesubsequent sample). Tenfold serial dilutions of lung homogenates were plated in

FIG. 1. Aerosol delivery system. The system, housed in a biosafetycabinet, allowed us to simultaneously expose up to 4 mice to aerosolpreparations. A small compressor transferred a bacterial suspension asan aerosol to each of the four inhalation chambers, each consisting ofa 30-ml syringe. The standard aerosol exposure cycle was as follows: 60min for nebulization (infection), 5 min for cloud decay, and UV irra-diation for 5 min (decontamination).

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triplicate on Mueller-Hinton agar (Oxoid SpA), and the number of colonies wascounted 24 h after incubation at 37°C. Bacterial colony counts from each lungwere normalized according to the wet weight of lung and then calculated asCFU/g, averaged, and compared between groups.

To assess bacterial dissemination, the spleen was also removed from eachmouse in each group at each time point and homogenized as described for thelungs. Serial dilutions were also plated and analyzed for colony counts. Thenumber of animals with a spleen positive for S. maltophilia was counted, andvalues were compared as percentages for each animal group and time point.

Cytokine and chemokine measurements. A protease inhibitor cocktail (Pierce,Rockford, IL) was added to the lung samples immediately after collection. Lunghomogenates were centrifuged at 1,500 � g at 4°C for 10 min, and then thesupernatants were removed, aliquoted, and stored at �80°C until their use. Thelevels of 9 cytokines (interleukin-1� [IL-1�], IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, gamma interferon [IFN-], and tumor necrosis factor alpha [TNF-]) and 9chemokines (keratinocyte-derived cytokine [GRO/KC], monocyte chemotacticprotein 1 [MCP-1/JE], macrophage chemoattractant protein 5 [MCP-5], macro-phage inflammatory protein 1 [MIP-1], MIP-2, regulated on activation, nor-mal T-cell expressed and secreted [RANTES], thymus- and activation-regulatedchemokine [TARC], eotaxin, and stromal cell-derived factor 1� [SDF-1�]) weresimultaneously measured in supernatants from lung homogenates by a multiplexsandwich enzyme-linked immunosorbent assay (ELISA) system based on chemi-luminescence detection (SearchLight chemiluminescent array kits; Endogen)according to the manufacturer’s recommendations. The cytokine and chemokinelevels were normalized according to the wet weight of lung tissue and arereported as pg/mg. The detection limits were 31.3 pg/ml (IFN-), 12.5 pg/ml (IL-1�and TNF-), 3.1 pg/ml (IL-2, IL-4, IL-10, and IL-12 p70), 6.3 pg/ml (IL-5), 21.9pg/ml (IL-6), 0.8 pg/ml (eotaxin), 1.6 pg/ml (MCP-5), 3.0 pg/ml (MIP-1, RANTES,and MCP-1/JE), 6.0 pg/ml (MIP-2, TARC, and GRO/KC), and 37.5 pg/ml(SDF-1�).

Statistical analysis. All analyses were conducted with GraphPad Prism 4.0software (GraphPad Software Inc., San Diego, CA). Differences between studiedgroups were evaluated using unpaired Student’s t test or analysis of variance(ANOVA) followed by Bonferroni’s multiple comparison posttest for parametricdata, the Kruskal-Wallis test followed by Dunn’s multiple comparison posttestfor nonparametric data, and the chi-square test for percentages. Survival of S.maltophilia-infected and control mice was compared by Kaplan-Meier survivalanalysis and the log rank test. With regard to cytokine and chemokine expres-sion, cluster analysis was performed by exporting logarithmic ratios of exposed tocontrol values at each time point for each cytokine/chemokine into Permut-Matrix software, version 1.9.3 (http://www.lirmm.fr/�caraux/PermutMatrix/). Hi-erarchical clustering was performed, using the Euclidean distance between pairsof observations and average linkages, to determine the degree of association(distance) between sets of observations. The results are presented as dendro-grams.

RESULTS

Mouse health and survival monitoring. The mean bodyweight before infection was 19.1 � 1.4 g for female mice and23.3 � 1.7 g for male mice. The changes in body weight ofinfected and control mice over time, expressed as percentagesof initial body weights, are shown in Fig. 3A. Mice infectedwith S. maltophilia lost more than 10% of their body weightfrom day 1 through day 7, and during this period the meanweight of infected mice was significantly (P � 0.01) lower thanthat of control mice. By day 5, infected mice slowly startedregaining weight, although they did not regain it completelyduring the period monitored. Control mice lost up to 5% oftheir body weight during the first 3 days and then startedgaining weight, with their weight regained completely on day 4.The infected mice usually showed symptoms of slow respon-siveness and piloerection from day 1 through day 6 and ap-peared healthy the next day.

The survival of DBA/2 mice was monitored over a period of14 days (Fig. 3B). Among 56 DBA/2 mice infected with S.maltophilia OBGTC9, a total of 12 mice died (Kaplan-Meiersurvival proportion of 59.4%): 7 mice died on day 3 (80.5%survival proportion), 4 mice died on day 4 (63.6% survivalproportion), and 1 mouse died on day 5 (59.4% survival pro-

FIG. 2. Experimental design. Groups of mice were exposed toaerosol with sterile PBS alone (control group; 6 mice/group) or con-taining S. maltophilia OBGTC9 (infected group; 12 mice/group). Onday 0 (1 h after exposure), the pulmonary bacterial load of DBA/2 mice(n � 8) was assessed. Mice were then sacrificed on days 1, 2, 3, 7, and14 after exposure for microbiological analysis of the lung and spleen,macroscopic description and histopathology of lungs, and measure-ment of pulmonary cytokines/chemokines. All mice were further mon-itored daily for survival and general health.

FIG. 3. (A) Weight monitoring during S. maltophilia lung infection.DBA/2 mice (n � 92) were exposed on day 0 to aerosolized S. malto-philia OBGTC9 (Œ) or PBS only (f) and were examined daily forweight loss during the course of infection. The dotted line shows a 10%weight loss with regard to mean body weight before infection. **, P �0.01 for control versus infected mice (unpaired Student’s t test). Errorbars represent SD. (B) Survival of DBA/2 mice after pulmonary infec-tion with S. maltophilia OBGTC9. Results are the combination of twoindependent experiments (n � 28 for uninfected mice; n � 56 forinfected mice) monitored over 14 days. The difference in survivalbetween infected and control mice was statistically significant (P �0.01; log rank test).

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portion). Mortality was not observed in control mice. Thedifference in survival between infected and control mice wasstatistically significant (P � 0.01).

Kinetics of bacterial burden in the lungs and dissemination.To explore the kinetics of S. maltophilia OBGTC9 infection inDBA/2 mouse lungs, bacterial loads were evaluated in lung tissuehomogenates on day 0 (1 h), 1, 2, 3, 7, and 14 after a singleexposure to S. maltophilia aerosol (Fig. 4A). The initial depositionof S. maltophilia observed after 1 h (day 0) was 4.9 � 107 � 3.0 �107 CFU/g. Bacterial loads decreased from day 1 to day 2, al-though not significantly (day 1, 5.2 � 107 � 3.9 � 107 CFU/g; day2, 1.5 � 106 � 1.5 � 106 CFU/g; P � 0.05). A statistically signif-icant decrease was observed from day 3 (2.3 � 105 � 2.9 � 105

CFU/g; P � 0.05 versus day 0 and day 1) through day 7 (3.5 �103 � 3.0 � 103 CFU/g; P � 0.01 versus day 0 and day 1). On day14, bacterial loads became undetectable in all infected mice. No S.maltophilia growth was observed at any time point in the lunghomogenates from control mice.

To evaluate the invasiveness of S. maltophilia, we carried outmicrobiological analysis of spleen homogenates (Fig. 4B).Overall, S. maltophilia was found in 59.1% (26 of 44 spleens) ofspleen samples tested, although the bacterial counts were gen-erally very low at each time point, i.e., 1 h (positive spleens, 6of 8 [75%]; bacterial load, 80 � 83 CFU), day 1 (10 of 11spleens [90.9%]; 7 � 6 CFU), day 2 (4 of 8 spleens [50%]; 4 �2 CFU), day 3 (1 of 10 spleens [10%]; 1 CFU), and day 7 (5 of7 spleens [71.4%]; 9 � 6 CFU) after exposure. The highestpercentage of S. maltophilia-positive spleens was observed onday 1 (P � 0.001), while no bacteria were isolated from spleenson day 14. Spleens from control mice were negative for S.maltophilia.

Macroscopic lung pathology. Macroscopic DBA/2 mouselung pathologies, assessed by using a four-point scoring system(19), are summarized in Fig. 5. More-severe changes in lungpathology were observed in infected mice on days 3, 7, and 14than on day 1 (P � 0.05). In particular, a median score of �1(normal lungs) was observed after 1 h and on day 1, a medianscore of �2 (swollen lung, hyperemia, and small atelectasis)was observed on day 2, the maximum score of �4 (large atel-ectasis and hemorrhages) was observed on day 3 (Fig. 5C), andfinally, a median score of �3 (pleural adhesion and atelectasis)was observed on days 7 and 14. Necropsy of mice that suc-cumbed to infection revealed a consolidated lung, marked

FIG. 4. Lung clearance and dissemination of S. maltophilia infec-tion. (A) DBA/2 mouse (n � 92) lung clearance kinetics after respi-ratory exposure to S. maltophilia strain OBGTC9. Lungs were col-lected, homogenized, and cultured for bacterial counts at 0 (1 h), 1, 2,3, 7, and 14 days postexposure. Results were normalized to the lungwet weight (CFU/g) and are shown as means � SD. *, P � 0.05; **,P � 0.01 versus day 0 (1 h) and day 1 (ANOVA [P � 0.0001] followedby Bonferroni’s multiple comparison posttest). (B) Percentages ofDBA/2 mice (n � 92) with spleens positive for S. maltophilia followinglung infection. Spleens were collected, homogenized, and cultured forbacterial counts after 0 (1 h), 1, 2, 3, 7, and 14 days. Results areexpressed as percentages of spleens positive for S. maltophilia at cul-ture analysis. ∧∧, P � 0.001 versus day 1, day 2, day 3, and day 14; **,P � 0.001 versus each time point; EE, P � 0.001 versus day 1, day 2,day 3, and day 14 (chi-square test).

FIG. 5. Macroscopic pathology of DBA/2 mouse lungs infectedwith S. maltophilia strain OBGTC9. (A) Macroscopic lung pathology inDBA/2 mice (n � 92) assessed on day 0 (1 h), 1, 2, 3, 7, and 14postexposure by use of a four-point scoring system proposed by Dubinand Kolls (19). Results are shown as follows: the line within each boxis the median; the upper and lower lines of the box are the 75th and25th percentiles, respectively; and the whiskers are the highest andlowest values. *, P � 0.05 versus day 1 (Kruskal-Wallis test [P � 0.01]followed by Dunn’s multiple comparison posttest). (B) Photograph ofuninfected mouse lung on day 3. (C) Photograph of S. maltophilia-infected mouse lung on day 3.



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edema, and hemorrhages. Control mice showed a score of �1(normal lungs) during the 14 days monitored (Fig. 5B).

Lung histopathology. The kinetics of lung histopathologyand sections illustrating lung inflammation during the study

period are shown in Fig. 6. Since there were no significantdifferences in the degree of inflammation at any of the timepoints by evaluating the left and right lungs separately, the datawere pooled and considered representative of both lungs.

FIG. 6. (A) Tissue histopathology of lungs of DBA/2 mice infected with S. maltophilia strain OBGTC9. Lung sections of DBA/2 mice (n � 92)were stained with Giemsa stain and are representative of eight (infected group) or four (control group) mice per group studied at each time point(1 h and 1, 2, 3, 7, and 14 days). Magnification, �20 (bronchial sections) and �10 (alveolar sections). (B) Microscopic DBA/2 mouse lung pathologyfollowing infection with S. maltophilia strain OBGTC9. Lung pathologies of DBA/2 mice (n � 92) were assessed by use of a five-point scoringsystem proposed by Johansen et al. (27). Results are shown as median values. *, P � 0.05; **, P � 0.01 for bronchial (f) versus alveolar (Œ)sections (Kruskal-Wallis test [P � 0.01] followed by Dunn’s multiple comparison posttest).



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In infected mice, we observed endobronchial inflammation,predominantly mononuclear, increasing from day 1 to day 3;thereafter, the infection moved toward the parenchyma, in-creased in inflammation score, involved alveolar walls and al-veolar sacs, in succession, and induced increasing of septalthickening, edema, obliteration of the majority of alveolarspaces, and fibrosis. In particular, for bronchial lumens ob-tained from day 1 through day 3, we observed a predominantlypolymorphonuclear infiltrate in 60% (day 1; score of 2), 30%(day 2; score of 3), and 45% (day 3; score of 3) of visualizedlumens. On day 7, there was no sign of inflammation in thebronchial lumen (score of 0).

On day 1, in the alveolar parenchyma, we observed an in-crease of interstitial cellularity and increased thickness of thealveolus-capillary barrier (score of 2). Starting from day 2,edematous bleeding and obliteration of an increasing propor-tion of alveolar ducts were evident, in 15% (day 2; score of 2)to 80% (day 14; score of 3) of cases.

Generally, lungs of untreated control animals had little or noevidence of inflammation in the airways or lung parenchyma.Necropsy of mice that succumbed to infection revealed markedto severe alveolar hemorrhages and numerous neutrophils inthe alveolar compartment.

Cytokine and chemokine levels in mouse lungs. To gaininsight into the difference between the immunological reac-tions of the infected and uninfected DBA/2 mice, we measuredthe levels of 9 cytokines and 9 chemokines in the lung homog-enates on days 1, 3, and 7. The expression levels of cytokines inthe lungs of S. maltophilia-infected and control mice are re-ported in Fig. 7. Statistically higher levels of the followingcytokines were observed in infected mice on day 1 than incontrols: IL-1� (9.6 � 4.1 pg/mg versus 2.9 � 0.7 pg/mg,respectively; P � 0.01), IL-6 (187.8 � 152.0 pg/mg versus 3.2 �1.2 pg/mg, respectively; P � 0.05), IL-12 (1.1 � 0.2 pg/mgversus 0.1 � 0.05 pg/mg, respectively; P � 0.01), IFN- (31.9 �12.6 pg/mg versus 2.3 � 1.0 pg/mg, respectively; P � 0.01), andTNF- (14.7 � 5.1 pg/mg versus 0.1 � 0.08 pg/mg, respectively;P � 0.01) (Fig. 7A). On day 3, cytokine levels in infected micewere also higher than those in controls, although the differ-ences were not statistically significant, with the exception ofIFN- (23.0 � 7.9 pg/mg versus 0.5 � 0.2 pg/mg, respectively;P � 0.01). IL-4 levels on day 1 were statistically higher incontrol than in infected mice (2.2 � 0.9 pg/mg versus 1.0 � 0.5pg/mg, respectively; P � 0.01). All other cytokines measuredwere not significantly different between infected and controlmice at the time points tested. Hierarchical cluster analysis ofcytokine levels, based on the log ratios for infected versuscontrol mice, revealed three different expression profiles: (i)TNF-, IL-6, and IFN-; (ii) IL-12 and IL-1�; and (iii) IL-4,IL-5, and IL-10 (Fig. 7B).

The expression levels of chemokines in the lungs of S. mal-tophilia-infected and control mice are represented in Fig. 8.Statistically (P � 0.01) higher levels of the following chemo-kines were observed in infected mice on day 1 than in controls:GRO/KC (514.8 � 316.8 pg/mg versus 1.0 � 0.4 pg/mg,respectively), MCP-1/JE (74.2 � 40.9 pg/mg versus 1.1 � 0.8pg/mg, respectively), MCP-5 (95.2 � 76.8 pg/mg versus 5.4 �2.3 pg/mg, respectively), MIP-1 (33.1 � 9.6 pg/mg versus3.1 � 1.5 pg/mg, respectively), MIP-2 (69.4 � 23.0 pg/mgversus 4.2 � 2.4 pg/mg, respectively), and TARC (64.5 � 20.5

pg/mg versus 30.8 � 13.9 pg/mg, respectively) (Fig. 8A). After3 and 7 days, chemokine levels in infected mice were alsohigher than those in controls, although the differences were notstatistically significant. All other chemokines measured werenot significantly different between infected and control mice atthe time points tested. Hierarchical cluster analysis of chemo-kine levels, based on the log ratios for infected versus controlmice, revealed two different expression profiles: (i) JE/MCP1,MIP-2, MIP-1, and MCP-5; and (ii) eotaxin, TARC, RAN-TES, and SDF-1� (Fig. 8B).

DISCUSSION

Robust experimental pulmonary infection models are a pre-requisite for the understanding of the pathogenicity of micro-organisms in the lung and the related host defense mecha-nisms. However, the route of administration is often associatedwith technical difficulties, especially in the case of small exper-imental animals such as mice.

Different techniques have been employed to introducepathogens into the lung, such as intratracheal instillation (10),intranasal application (54), and application via aerosol (55,56). Intratracheal administration allows the colonization of thelung by a large fraction of the inoculum, with a potentially lowlevel of extrapulmonary contamination. However, the immo-bilization of bacteria on agar beads is time-consuming andcould be associated with a low level of reproducibility in bac-terial lung load, probably because of its difficult standardiza-tion. Furthermore, the trauma caused by the surgery involvedin the tracheotomy often results in moribundity or death ofanimals.

The intranasal instillation and aerosol administration meth-ods require little experimental effort and are the least invasivemethods. However, low reproducibility and contamination ofthe lung by nasal and oropharyngeal flora are the major dis-advantages of nasal instillation. In contrast, administration viaaerosol by our inhalation system ensures high reproducibility(both between different experiments and between animals in agiven experiment) and no lung contamination, and it is nottime-consuming because it permits infection of up to fouruntrained mice simultaneously. Furthermore, we also assessedthat bacteria are not lysed by shear forces when pressedthrough the nebulizer, thus not causing exposure to a highantigen load (data not shown). In contrast to tracheal surgery,the use of aerosolization as the method of exposure also avoidsthe use of anesthetic agents, which are known to affect pulmo-nary clearance (23), and permits the exclusion of surgery-re-lated inflammation while trying to study the inflammatory re-sponse to bacterial infection. Lastly, the aerosol deliverytechnology simulates the natural route for acquiring deep-lunginfections in humans.

Both acute and chronic models of lung infection have beenestablished in several animal species, including rats, guineapigs, hamsters, mice, mink, sheep, rabbits, and monkeys (48).The DBA/2 inbred mouse strain, a model already used for thestudy of pathogenesis in CF (4, 7, 36, 45), is characterized by anautosomal recessive mutation of the gene controlling the he-molytic activity of the serum, resulting in a deficiency in theproduction of the C5 component. This component participatesin the host defense against infection and in the inflammatory



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FIG. 7. Time course expression of cytokines in control and infected lungs. (A) Cytokine levels measured on days 1, 3, and 7 postexposure inlung homogenates from control (white bars) and infected (gray bars) mice (n � 54). Results were normalized to the lung wet weight (pg/mg) andare shown as means plus SDs. Statistically significant (*, P � 0.05; **, P � 0.01 [ANOVA followed by Bonferroni’s multiple comparison posttest])differences were observed in the levels of protein expression between infected and control mice for IL-1�, IL-4, IL-6, IL-12, IFN-, and TNF-.(B) Hierarchical clustering expression plot. Different colors in the rectangles represent the average log ratios, defined as log2 (infected value/control value) for each cytokine. The dendrogram illustrates the degrees of similarity in expression between the cytokines tested. The color barbeneath the dendrogram represents the logarithmic expression values.



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FIG. 8. Time course expression of chemokines in control and infected lungs. (A) Chemokine levels measured at 1, 3, and 7 days postexposurein the lung homogenates from control (white bars) and infected (gray bars) mice (n � 54). Results were normalized to the lung wet weight (pg/mg)and graphed as means plus SD. Statistically significant (**, P � 0.01 [ANOVA followed by Bonferroni’s multiple comparison posttest]) differenceswere observed in the levels of protein expression between infected and control mice for GRO/KC, MCP-1/JE, MCP-5, MIP-1, MIP-2, andTARC. (B) Hierarchical clustering expression plot. Different colors in the rectangles represent the average log ratios, defined as log2 (infectedvalue/control value) for each chemokine. The dendrogram illustrates the degrees of similarity in expression between the chemokines tested. Thecolor bar beneath the dendrogram represents the logarithmic expression values.



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response by means of multiple biological activities, such aschemoattraction, stimulation of phagocytes to release cyto-kines (TNF- and IL-1), granule enzymes, and oxygen metab-olites, and enhancement of antibody formation (28).

For these reasons, in the present study we used aerosoldelivery technology for the deposition of S. maltophilia intoDBA/2 mouse lungs. Using this model of respiratory infection,we investigated bacterial clearance, histopathology, and theinflammatory response in the mouse lungs following a singleexposure to aerosolized S. maltophilia.

Unlike chronic models using agar beads that hold the bac-teria in the airway by mechanical means, thus bypassing initialbacterial attachment and retention, our acute model requiresS. maltophilia suspended in PBS, thus allowing us to test theability of the animals to clear bacteria from the lung and toestablish the relative importance of antibacterial factors inearly development of pulmonary disease in CF. Our resultsshowed that almost all (�99.99%) of the organisms initiallydeposed in the lung were killed within the first 7 days afterinoculation, thus greatly reducing the inciting stimulus. On day14, bacterial loads became undetectable in all infected DBA/2mice, suggesting that they were able to resolve S. maltophilialung colonization in a period ranging from 8 to 14 days.

The clearance of S. maltophilia from the lungs of DBA/2mice was clearly associated with the profile of the inflamma-tory response observed in the DBA/2 lung tissue, which con-sisted of an early and intense, but detrimental, bronchial andalveolar inflammatory response, mediated primarily by neutro-phils—the prominent inflammatory cell in the lungs of CFpatients, even in individuals with minimal pulmonary involve-ment (32)—followed shortly by an influx of inflammatory mac-rophages.

Since DBA/2 mice are one of several C5-deficient inbredmouse strains (55), thus predicting a reduction in neutrophilrecruitment to the lung tissues, the increased influx of inflam-matory leukocytes we observed suggested that the loss of C5amay be compensated by the host by increasing the productionof redundant/alternative neutrophil chemotactic factors.

In the present study, we evaluated the magnitude of theinflammatory response provoked by S. maltophilia exposure bymeasuring the DBA/2 mouse pulmonary levels of 9 cytokinesand 9 chemokines, selected on the basis of the specific proin-flammatory protein profiles found in infected CF patients (6,11, 26, 31, 33).

In agreement with what has been observed in lung secretionsfrom patients with CF with respect to healthy controls (6), wefound statistically (P � 0.01) higher levels of TNF-, IL-1�,and IL-6 in infected mice than in controls.

TNF- has been implicated in the inflammation in and clear-ance of P. aeruginosa from the murine lung (39). TNF- con-tributes to the restriction of microbial growth by upregulatingadhesion molecules, such as ICAM-1, involved in polymorpho-nuclear leukocyte (PMN) recruitment and by amplifying theinnate clearance mechanisms (40). Waters et al. (54) recentlyfound that S. maltophilia induced substantially more TNF-expression by macrophages than did P. aeruginosa, probablydue to the high degree of lipid A heterogeneity.

IL-6 is constitutively upregulated in CF patients, leading toincreased neutrophil recruitment and further enhancement ofinflammation in the lung (16, 26).

We also found significantly higher IFN- and IL-12 levels ininfected lungs than in controls. The inflammatory cytokineIFN- is produced by T lymphocytes and natural killer (NK)cells and is able to activate the microbicidal function of mac-rophages and NK cells (5). Furthermore, IFN- can enhancethe chemotaxis and phagocytosis of PMNs to pathogens (37).We found that IFN- was the only cytokine with persistenthyperexpression, since levels in infected mice were significantlyhigher than those in controls until day 3.

IL-12 (IL-12 p70) is secreted mainly by peripheral lympho-cytes after bacterial induction and stimulates production ofIFN- by NK and T cells (5). Although IL-12 is essential forthe host defense against various pathogens, this cytokine is notlikely to be a major player in the host response to P. aeruginosalung infection (42).

The increases in IFN- and IL-12 were in agreement withthe decrease, though not statistically significant, of IL-10 inlung tissues, since IL-10 suppresses the synthesis of proinflam-matory cytokines by inhibiting IL-12 and IFN- production (5).In addition, we observed that levels of the pleiotropic cytokineIL-4, a product of Th2 lymphocytes that inhibits the differen-tiation of Th1 cells (5, 24), were also reduced, even though thisdifference was not statistically significant.

Among chemokines, statistically (P � 0.01) higher levelswere observed in infected mice on day 1 than in controls forGRO/KC, MCP-1/JE, MCP-5, MIP-1, MIP-2, and TARC.GRO/KC and MIP-2—murine homologues of human IL-8known to be expressed differently in the lungs of CF patients(16, 26, 33)—are overexpressed in murine keratinocytes,monocytes, and macrophages and are involved in chemotaxisand cell activation of neutrophils in the mouse (44). MIP-1,produced by macrophages following their stimulation with bac-terial endotoxins and involved in the cell activation and re-cruitment of neutrophilic granulocytes, is elevated in the lungsof young children with CF, even in the absence of pulmonaryinfection (8). MCP-5 and MCP-1/JE are potent chemoattrac-tants for peripheral blood monocytes only, while increasedlevels of TARC suggest a T-cell chemoattraction and adhesionof monocytes to the endothelium.

Taken together, our results suggest that the immune re-sponse to acute infection with S. maltophilia is predominantlya Th1-type response characterized by the recruitment of PMNsto the lung tissue as a result of the excessive production ofTNF-, IL-1�, and the PMN chemoattractants MIP-1,MIP-2, and GRO/KC. Similarly, higher levels of MCP-1/JEand MCP-5 are responsible for monocyte recruitment.

An indirect indicator of the failure of mice to control aGram-negative infection is weight loss due to the induction ofcachexia by LPS-induced inflammatory cytokines. Cachexiaobserved in CF patients might be the outcome of excessiveconcentrations of TNF- (20). Interestingly, our resultsshowed that the timing of the most severe mouse weight lossimmediately followed the most dramatic difference in TNF-concentration, thus confirming that it is a pivotal proinflam-matory cytokine that plays an important role in inducing theexcessive inflammatory response in the CF lung, as well asadditional systemic effects.

The potential of S. maltophilia to cause invasive infectionshas been reported previously (35, 38). Recently, we showedthat S. maltophilia is able to invade cultured A549 respiratory



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epithelial cells, although at very low levels (18). The resultsfrom the present study confirm the scant invasiveness of S.maltophilia, as suggested by a transient and minimal presenceof the microorganism in the spleens of DBA/2 mice. In agree-ment with the work of Waters et al. (54), it is plausible thatduring pulmonary infection, the few bacteria crossing the ep-ithelial barrier are readily cleared, if not by lytic effects ofserum, then by phagocytosis, and they do not produce sufficientconcentrations in the blood to provoke sepsis. The sequencedS. maltophilia genome has few regions with low levels of ho-mology to any of the P. aeruginosa type III secretion genes(12). Type III secretion systems mediate bacterial interactionswith host cytoskeletal components in many Gram-negativepathogens, and in P. aeruginosa, they highly correlate withinvasive infection (25). Thus, the potential lack of type IIIsecretion genes in S. maltophilia may contribute to its limitedinvasive capabilities.

S. maltophilia generally causes infections that result in in-creased morbidity, but not usually in mortality, in the immu-nocompromised host. Our results showed that excessive pul-monary infection and inflammation had systemic effects,manifested by weight loss, and ultimately caused a large num-ber of deaths of infected mice compared to the control ani-mals. The high overall mortality rate we observed in thepresent study indicates the severity of S. maltophilia infection,as confirmed by the macroscopic in situ lung analysis, revealingedema, hemorrhage, atelectasis, consolidated areas, and fibrin-ous adhesion to the thoracic wall. Contrarily to our findings,Waters et al. (54) found that S. maltophilia CF strains causedno mortality in a neonatal mouse model of respiratory tractinfection.

While infection disseminated, as suggested by the presenceof bacteria in the spleens of DBA/2 mice, the death of miceappeared to be due primarily to the complication in the lungs,indicating that the DBA/2 mice were unable to overcome theincreased and prolonged inflammatory response provoked byS. maltophilia infection.

In conclusion, our results taken together show that the lungsof DBA/2 mice undergoing a single aerosol exposure to S.maltophilia in suspension share many characteristics with thepulmonary disease in CF patients: (i) the lung contains a largenumber of neutrophils, the prominent inflammatory cells in CFlungs; (ii) the remarkably high levels of proinflammatory me-diators, especially chemoattractant cytokines, in the lungs aresimilar to those observed in infants and young children withCF, even when controlled for bacterial burden (1, 3, 6, 41),which may perpetuate the robust inflammatory response; and(iii) additional CF-like systemic effects, such as weight loss andmortality, were also observed.

By using our model, this study provides a framework forunderstanding the role that S. maltophilia plays in pulmonaryCF disease. The severe pathology and high mortality we ob-served in infected animals contrast with the view that S. mal-tophilia may be just a bystander in CF patients, supporting theidea that this opportunistic pathogen has the potential to con-tribute to the inflammatory process responsible for the dete-rioration of lung function in CF patients.

Since S. maltophilia can often be found in CF lungs togetherwith other CF pathogens, in particular P. aeruginosa (43), fur-ther studies will be needed to assess the contribution of S.

maltophilia to worsening the inflammation resulting from in-fection with other pathogens.

ACKNOWLEDGMENTS

We thank Paola Ascione (Department of Oncology and Neuro-sciences, G. d’Annunzio University of Chieti-Pescara, Chieti, Italy) forher technical help in preparing lung sections for histological analysis.We also thank Andreina Santoro for contributing to the revision of themanuscript.

This work was supported in part by the Fondazione Italiana per laRicerca sulla Fibrosi Cistica (grant FFC#7/2007) and in part by theMinistero Italiano dell’Universita e della Ricerca Scientifica (MIUR)(grant PRIN2007; protocol 2007LXNYS7).

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