Journal of Infection and Public Health (2009) 2, 136—140

Evaluation of interleukin-10 production inPseudomonas aeruginosa induced acutepyelonephritis

Rahul Mittal a,b,∗, Saroj Sharmab, Sanjay Chhibberb, Kusum Harjaib

a Division of Infectious Diseases, Childrens Hospital Los Angeles, Los Angeles, CA, USAb Department of Microbiology, Panjab University, Chandigarh, India

Received 25 May 2009; received in revised form 9 June 2009; accepted 13 June 2009

KEYWORDSIL-10;Pseudomonasaeruginosa;Biofilms;Pyelonephritis

Summary Pseudomonas aeruginosa is an opportunistic pathogen of immunocom-promised hosts. This pathogen has a tendency to form biofilms on the surface ofindwelling catheters leading to acute and chronic urinary tract infections that resultin significant morbidity and mortality. In the present study, kinetics of interleukin-10(IL-10) production in mouse renal tissue was studied employing experimental mousemodel of acute pyelonephritis induced with planktonic and biofilm cells of P. aerug-inosa. IL-10 production was found to be significantly lower in biofilm cell instilledmice compared to planktonic cell infected animals, which corroborated with higherbacterial load and tissue damage. The data suggests that downregulation of IL-10production may be novel strategy employed by biofilm cells to cause tissue dam-

age and hence bacterial persistence. The results of the present study may open upavenues of research that will ultimately provide the foundation for the developmentof preventative measures and therapeutic strategies to successfully treat P. aerug-inosa biofilm infections based on the administration of anti-inflammatory agents.

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Introduction

Urinary tract infections (UTIs) are one of themost common bacterial infections affecting humansthroughout their life span. UTIs account for about

∗ Corresponding author at: Division of Infectious Diseases,MS#51, Childrens Hospital Los Angeles, 4650 Sunset Boulevard,Los Angeles, CA, 90027, USA. Tel.: +1 323 361 5809.

E-mail addresses: ramittal@chla.usc.edu,mittal05@gmail.com (R. Mittal).

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1876-0341/$ — see front matter © 2009 King Saud Bin Abdulaziz University for Health

doi:10.1016/j.jiph.2009.06.002

ziz University for Health Sciences. Published by Elsevier

.3 million doctor visits each year at a cost of over1 billion [1]. Up to 40% of women will develop UTIt least once during their lives, and a significantumber of these women will have recurrent UTIs.seudomonas aeruginosa is the third most com-on pathogen causing hospital acquired catheter

ssociated UTIs [2]. Despite the use of antibiotic

herapy, the mortality and morbidity associatedith P. aeruginosa induced acute pyelonephritis

till remains high. This poor outcome is due tour incomplete understanding about the patho-

Sciences. Published by Elsevier Ltd. All rights reserved.

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L-10 and pyelonephritis

enesis of the disease. A large number of studiesave been performed targeted at the bacterialiofilms. However, little attention has been paido P. aeruginosa biofilms on urinary catheters.part from catheter removal, there is currently noreatment proven successful for completely erad-cating a biofilm-related infection caused by P.eruginosa, highlighting the need for research inhis area. Transplantation procedures, immunosup-ression and the use of urinary catheters havencreased the prevalence of P. aeruginosa inducedyelonephritis. Formation of biofilms on urinaryatheters offers survival advantage to P. aerugi-osa [3]. Biofilm infections are important clinicallyecause bacteria in biofilms exhibit recalcitrance tontimicrobial compounds and persistence in spite ofustained host defenses [4—6].

The interplay between the proinflammatory andnti-inflammatory mechanisms/mediators is essen-ial in fight of the host against the invadingathogen in limiting its effect and resolving inflam-atory process. In this context, interleukin-10 (IL-

0), an anti-inflammatory cytokine, appears rela-ively late following induction of immune responses7]. This cytokine has been reported to be producedy T cells, B cells and macrophages/monocytes [8].L-10 pathway is a critical regulator of immuneesponses to microorganisms. The challenge facedy the immune system of an infected host is toespond with sufficient intensity and duration toontrol and eliminate the infection while mini-izing nonspecific injury to host tissue. Within

he context of infectious disease, IL-10 plays anmportant role in the balance between protec-ive immunity and the development of immuneathology. IL-10 modulates expression of cytokinesnd soluble mediators by immune cells like mono-ytes, with important consequences for their abilityo activate and sustain immune and inflamma-ory responses [9]. However, the role of IL-10n UTIs has not been elucidated. In the presentnvestigation, we studied the production of IL-10uring P. aeruginosa induced acute pyelonephritisnd its correlation with bacterial load and tissueamage. The results of this study may provide aindow of opportunity for the potential use of anti-

nflammatory intervention against UTIs.

ethods

rganisms

urinary isolate of P. aeruginosa, employed in ear-ier studies [10—12], isolated from a hospitalizedatient having complicated UTI and serotyped as

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137

11 by the Laboratory of HealthCare Associatednfection, London was used. In addition, a stan-ard strain of P. aeruginosa, PAO was obtained fromr. Barbara H. Iglewski, University of Rochester,ochester, New York. Both the strains were grownvernight in nutrient broth at 37 ◦C and harvestedy centrifugation. Cells were washed three timesith phosphate-buffer saline (PBS), resuspended

o a concentration of 1 × 108 organisms per ml inBS (confirmed by viable cell counting) and weresed as planktonic cells for induction of infectionn mice.

eneration of biofilm cells

he method of Mittal et al. [10] was followedor generation of biofilms. Both the uroisolate andAO strain of P. aeruginosa were used to generateiofilm cells. Foley’s catheter (Bardia) was cut into.0 cm pieces, inoculated with 100 �l of overnightrown culture and incubated at 37 ◦C. Every 24 hatheter pieces were removed from each flask,insed three times with PBS and transferred to theew flask containing fresh medium until day 4. Onay 4, catheter pieces were rinsed three times withBS to remove adherent bacteria and sliced lon-itudinally into equal halves. Cells were removedrom the surface of catheter pieces by scrappinghe inner surface with a sterile scalpel blade. Theispersed sample was then centrifuged, and theiofilm cells were suspended in 1 ml PBS. Bacterialoncentration was confirmed by viable counts.

nduction of ascending pyelonephritis

or induction of ascending pyelonephritis, femaleACA mice, 6—8-week-old, weighing 25 ± 5 gbtained from Central Animal House, Panjabniversity, Chandigarh, India were used. Animalsere kept in clean polypropylene cages underathogen-free conditions and given food and waterd libitum. The bladder of animals was pressedo evacuate urine before instillation. Urine washecked for preinfection bacteriuria by culturingn selective media. A soft intramedic polyethy-ene catheter, non-radiopaque (outer diameter.61 mm, Clay Adams, USA) was inserted intohe bladder through the urethral meatus, and.05 ml of inoculum containing 108 CFU/ml waslowly injected into the bladder to avoid leakage10—12]. The catheter was kept in place for 10 min

fter completion of instillation and then it wasithdrawn carefully. No obstruction or furtheranipulation of the urinary tract was done. 8ice were used for each strain and for each time

nterval separately for planktonic and biofilm cell

138 R. Mittal et al.

Figure 1 (A) IL-10 levels in renal tissue of mice infected with planktonic and biofilm cells of uroisolate and standardissuefilm

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strain (PAO) of P. aeruginosa. (B) Bacterial load in renal thigher whereas IL-10 levels were significantly lower in bioanimals (P < 0.001, by Student’s t-test).

forms. All animal experiments were carried out intwo groups in triplicate. The study protocol wasapproved by the Institutional Ethical Committeefor Animal Experimentation.

Bacteriological examination

Animals were sacrificed at 1, 3, 5, 7, 10 and 14 dayspost-infection. One-half of both the renal tissuewas removed aseptically, weighed and homoge-nized in 1 ml of sterile PBS. This homogenized tissuewas plated on cetrimide agar plates. Quantitativebacterial counts per gram of the renal tissue werecalculated [10].

Histopathological examination

The other half of the renal tissue was fixed in10% buffered formal saline and was dehydrated inethanol gradient of 30—100%. Tissues were thenembedded in wax, sectioned and stained withhemotoxylin and eosin (H&E) [11].

Cytokine response

IL-10 levels in renal tissue were quantitated byusing an ELISA kit (R&D Systems, Minneapolis).ELISA was carried out according to the manufac-turer’s instructions.

Assessment of neutrophil response

Tissue neutrophils were quantitated using a

myeloperoxidase (MPO) assay [10]. Renal tissuewas homogenized in 2 ml of 50 mM potassiumphosphate, pH 6.0 with 5% hexadecyltrimethylam-monium bromide and 5 mM EDTA. Homogenateswere sonicated and centrifuged. Supernatants were

itacm

of pyelonephritic mice. Bacterial load was significantlycell instilled mice compared to planktonic cell infected

ixed at the ratio of 1:15 with assay buffer and readt 490 nm. MPO units were calculated as the changen absorbance over time.

alondialdehyde (MDA) estimation

DA was estimated following method of Wills [13].riefly, tissue supernatant was added to equalmount of Tris—HCl (0.1 M, pH 7.4) and incubatedt 37 ◦C for 2 h. After incubation, trichloroaceticcid was added and centrifuged at 700 × g for0 min. Supernatant was mixed with equal volumef thiobarbituric acid (0.67%, w/v) and kept in boil-ng water bath for 10 min. After cooling, volumeas made to 3 ml with double distilled water andbsorbance was taken at 532 nm. Amount of MDAormed was expressed in nanomoles per milligramrotein (nmol/mg) using an extinction coefficientf 1.56 × 105 M−1 cm−1.

tatistical analysis

or statistical analysis of data, ANOVA, Fischer testnd Student’s t-test was applied, and P values werealculated. P < 0.05 were considered statisticallyignificant.

esults and discussion

n the present study, kinetics of IL-10 produc-ion was studied in P. aeruginosa induced acuteyelonephritis employing mouse model of ascend-

ng urinary tract infection. IL-10 production startedo appear in kidneys on 3rd post-infection daynd reached a peak on 7th post-infection day inase of both planktonic and biofilm cell infectedice (Fig. 1A). Thereafter gradual decrease in

IL-10 and pyelonephritis 139

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igure 2 (A) MPO levels (a marker of neutrophil influx)issue damage) in renal tissue of infected mice. MPO andice compared to planktonic cell infected animals (P < 0

roduction of IL-10 was observed till 14th post-nfection day. IL-10 levels were significantly lowern biofilm cell instilled mice compared to planktonicell infected animals (P < 0.001). Maximal produc-ion of IL-10 was accompanied with decrease inenal bacterial load leading to resolution of infec-ion by 14th post-infection day in planktonic cellnfected mice. However biofilm cells were able toersist in the renal tissue till 14th post-infectionay (Fig. 1B). Significantly lower levels of IL-10orroborated with higher neutrophil recruitmentassessed by MPO assay) (Fig. 2A) and hence tissueamage (assessed in terms of MDA assay) (Fig. 2B)n mice infected with biofilm cells as comparedo planktonic cell instilled mice. Histopathologi-al evaluation of the renal tissue revealed severenflammation along with destruction of tubules iniofilm cell infected mice (Fig. 3A). In contrast

ild inflammation was observable in renal tissue oflanktonic cell infected mice (Fig. 3B). In this con-ext, IL-10 has been reported to be an importantegulator of host response with a complex role in

atct

igure 3 Histopathological evaluation of renal tissue. Sevef biofilm cell instilled mice (A). In contrast, mild inflammanfected mice (B).

nal tissue of infected mice. (B) MDA levels (a marker ofA levels were significantly higher in biofilm cell instilled

by Student’s t-test).

odels of P. aeruginosa infection. IL-10 deficiencyas found to exacerbate lung damage whereas

reatment with IL-10 was associated with atten-ation of excessive inflammation in mouse modelf chronic endobronchial P. aeruginosa infection14]. These workers suggested use of IL-10 as aotential therapeutic agent for treatment of cys-ic fibrosis patient. Chmiel et al. [15] demonstratedrolonged inflammation, persistence of neutrophilsnd tissue damage in IL-10 deficient mice as com-ared to wild type mice following respiratory tractnfection with P. aeruginosa. However, no studiesre available regarding role of IL-10 in P. aerug-nosa induced acute pyelonephritis. The findingsf present study as well as available informa-ion from literature brings out that IL-10 has aotential for dampening inflammation resulting inesolution of urinary tract infections. The data

lso suggests that down-regulation of IL-10 produc-ion may be a novel strategy employed by biofilmells to persist inside host by causing inflamma-ion and hence tissue damage. This may help in

re inflammation (arrow) was observable in renal tissuetion (arrow) was seen in renal tissue of planktonic cell

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Crit Care Med 1999;160:2040—7.

140

forming a nidus where pathogen can grow andmultiply leading to recurrent and chronic infec-tions. This needs to be addressed to in futureinvestigations especially with different strains ofP. aeruginosa and other uropathogens which mayhelp in developing alternative preventive strategiesagainst pyelonephritis based on administration ofanti-inflammatory agents like IL-10.

Conflict of interest

Funding: This work was supported by a grantfrom Indian Council of Medical Research (ICMR),New Delhi, India.

Competing interests: None declared.Ethical approval: The study protocol was

approved by the institutional ethical committee foranimal experimentation.

Acknowledgements

We are grateful to Dr. Barbara H. Iglewski for provid-ing us with a standard strain of P. aeruginosa. Thiswork was supported by a grant from Indian Councilof Medical Research (ICMR), New Delhi, India.

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