mir-155 suppresses bacterial clearance in pseudomonas

M A J O R A R T I C L E

miR-155 Suppresses Bacterial Clearance inPseudomonas aeruginosa–Induced Keratitis byTargeting Rheb

Kun Yang,1,2,a Minhao Wu,1,2,a Meiyu Li,1,2 Dandan Li,1,2 Anping Peng,1,2 Xinxin Nie,1,2 Mingxia Sun,3 Jinli Wang,1,2

Yongjian Wu,1,2 Qiuchan Deng,1,2 Min Zhu,1,2 Kang Chen,1,2 Jin Yuan,3 and Xi Huang1,2,3

1Department of Immunology, Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 2Key Laboratory ofTropical Diseases Control, Ministry of Education and 3State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University,Guangzhou, China

miR-155 (microRNA-155) is an important noncoding RNA in regulating host inflammatory responses. However,its regulatory role in ocular infection remains unclear. Our study first explored the function of miR-155 in Pseu-domonas aeruginosa–induced keratitis, one of the most common sight-threatening ocular diseases. We found thatmiR-155 expression was enhanced in human and mouse corneas after P. aeruginosa infection and was mainly ex-pressed in macrophages but not neutrophils. In vivo studies demonstrated that miR-155 knockout mice displayedmore resistance to P. aeruginosa keratitis, with a higher inducible nitric oxide synthase level and a lower bacterialburden. More importantly, in vitro data indicated that miR-155 suppressed the macrophage-mediated bacterialphagocytosis and intracellular killing of P. aeruginosa by targeting Rheb (Ras homolog enriched in brain). Tothe best of our knowledge, this is the first study to explore the role of miR-155 in bacterial keratitis, which mayprovide a promising target for clinical treatment of P. aeruginosa keratitis and other infectious diseases.

Keywords. miR-155; Pseudomonas aeruginosa; corneal infection; phagocytosis; bacterial killing; inflammation;Rheb.

Pseudomonas aeruginosa is one of the bacterial speciesmost commonly isolated from contact-lens userswith corneal infection (keratitis) [1]. P. aeruginosa–induced keratitis is a suppurative ocular infectiousdisease that progresses rapidly and often leads to in-flammatory epithelial edema, stromal infiltration, tissuedestruction, and corneal ulceration and sometimesleads to vision loss [1].

Invading P. aeruginosa replicates within the hostbody and produces a variety of virulence factors, suchas exotoxin A [2], endotoxin lipopolysaccharide [3],and exoenzyme ExoU [4]. These virulence factors notonly induce the death of host cells by destroying the

plasma membrane or preventing protein synthesis,but also initiate the host inflammatory response,which may result in immunopathological tissue damage[1, 5, 6]. Once the bacteria break the anatomical barrier,inflammatory cells such as polymorphonuclear neutro-phils (PMNs) and macrophages are quickly recruited tothe infection site to engulf invading microorganisms[1]. Activated macrophages and PMNs produce alarge amount of reactive oxygen species and reactive ni-trogen species to kill the engulfed bacteria, thereby con-trolling the bacterial burden in the infected cornea.

Experimental P. aeruginosa challenge usually inducescorneal perforation in susceptible B6mice (T-helper type1 responders) at 5 days after infection, but disease ismuch less severe in resistant BALB/c mice (T-helpertype 2 responders) [7], indicating the importance of im-mune regulation in determining the disease outcome. Re-cently, several microRNAs have been implicated ascritical regulators in ocular diseases, such as miR-132in herpes simplex virus–induced keratitis and cornealneovascularization [8], miR17-92 cluster in retinoblasto-ma cell proliferation and invasion [9], and miR-155 in

Received 15 September 2013; accepted 31 December 2013; electronically pub-lished 7 January 2014.

aK. Y. and M. W. contributed equally to this article.Correspondence: Xi Huang, MD, PhD, Sun Yat-sen University Zhongshan School

of Medicine, Guangzhou 510080, China ([email protected]).

The Journal of Infectious Diseases 2014;210:89–98© The Author 2014. Published by Oxford University Press on behalf of the InfectiousDiseases Society of America. All rights reserved. For Permissions, please e-mail:[email protected]: 10.1093/infdis/jiu002

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experimental autoimmune uveitis and expansion of pathogenicT-helper type 17 cells [10]. However, nothing is known regardingthe role of microRNAs in modulating bacterial keratitis.

As one of the most studied microRNAs in the field of im-mune regulation, miR-155 executes multiple functions by tar-geting distinct messenger RNAs. Studies have demonstratedthat miR-155 enhances T-regulatory cell and T-helper type 17cell differentiation and T-helper type 17 cell function by target-ing SOCS1 (suppressor of cytokine signaling 1) [11], and atten-uates T-helper type 2 cell responses by targeting c-Maf, a potenttransactivator of the interleukin 4 promoter [12]. It is alsoreported that miR-155 enhanced proinflammatory cytokine re-sponse to Francisella novicida infection by targeting SHIP-1(Src homology-2 domain-containing inositol 5-phosphatase1) [13] but functions as a negative regulator of inflammationin endotoxin lipopolysaccharide–challenged human dendriticcells, by targeting TAB2 (transforming growth factor β–activatedkinase 1/MAP3K7 binding protein 2) [14, 15]. Nonetheless,whether miR-155 is involved in P. aeruginosa clearance andits downstream target remain unclear.

In the present study, we investigated the potential role ofmiR-155 in P. aeruginosa keratitis. Our study demonstrated thatmiR-155 expression was significantly induced after P. aeruginosainfection in vivo and in vitro, and contributed to corneal sus-ceptibility by suppressing bacterial eradication. More impor-tantly, we identified Rheb (Ras homolog enriched in brain) asa novel functional target of miR-155 in attenuating macro-phage-mediated bacterial phagocytosis and intracellular killing.These data may provide a promising therapeutic target for thetreatment of bacterial keratitis and other infectious diseases.

METHODS

Ethics StatementsFor studies using clinical human materials, written informedconsent from participating patients and approval from the Re-search Ethics Committee of Sun Yat-sen University (Guang-zhou, China) were obtained. For animal studies, all of theexperiments were performed in accordance with the guidelinesprovided in the Association for Research in Vision and Oph-thalmology statement for the Use of Animals in Ophthalmicand Vision Research and were approved by the Scientific Inves-tigation Board of Sun Yat-sen University.

ReagentsP. aeruginosa strain 19660 was from ATCC. Control and miR-155 mimics were from Applied Biosystems (Life Technologies,Grand Island, NY). LNA control and LNA miR-155 inhibitorswere from Exiqon (Vedbaek, Denmark). Anti-Rheb antibodywas from Abcam (New Territories, Hong Kong). Anti-SHIP1antibody was from Cell Signaling Technology (Beverly, MA).TaqMan microRNA assay kits were from Applied Biosystems.

Patients and Tissue SpecimensPatients with P. aeruginosa keratitis treated at the Sun Yat-senUniversity Zhongshan Ophthalmic Center (Guangzhou, China)between January 2011 and December 2012 were candidates forstudy inclusion. Criteria for inclusion were clinical diagnosis ofP. aeruginosa keratitis, with experimental confirmation by micro-bial culture of corneal scrapings. Patients were divided into 3groups of 8 patients on the basis of the time of corneal scrapingscollection: group 1, collection 1–6 days after infection (5 malesand 3 females; age range, 27–58 years); group 2, collection 7–13days after infection (5 males and 3 females; age range, 22–61years; and group 3, collection 14–30 days after infection (5males and 3 females; age range, 22–56 years). Corneal scrapingswere collected before the first treatment. Controls were normalcorneal tissues remaining after corneal transplantation, and con-firmed to be free of any prior pathologically detectable conditions.

Ocular InfectionEight-week-old female wild-type (WT) or miR-155 knockout(KO) C57BL/6 (B6) mice were from Jackson Laboratory (BarHarbor, ME). Infection of mouse corneas and scoring of corne-al disease were performed following the protocol reported else-where [16, 17], as described in the Supplementary Materials.

Plate Count to Assess Phagocytosis and Intracellular KillingPhagocytosis and intracellular killing were assessed by platecount, as described by others [4]. Cells were challenged withP. aeruginosa at a multiplicity of infection of 25. The numbersof internalized and killed bacteria were assessed after 1 or 2hours of incubation. After 1 hour, gentamicin was added tothe medium at 300 µg/mL for 30 minutes to kill extracellularbacteria. Cells were then lysed, and bacterial colony-formingunits (CFU) were determined by a plate count assay. Thephagocytosis efficiency was calculated on the basis of CFUdata obtained 1 hour after infection and was normalized tothe control group. A second series of internalization assayswas run in parallel to determine the number of viable bacteriafollowing 2 hours of incubation. For this, after the same treat-ment to remove extracellular bacteria, cells were incubated foranother 1 hour and then lysed for analysis of intracellular bac-terial CFU. The killing efficiency was calculated as [CFU (1hour) – CFU (2 hour)]/CFU (1 hour) and normalized to thecontrol group.

Statistical AnalysisThe differences in clinical score between 2 groups at each timepoint were tested by the Mann–WhitneyU test. For other exper-iments, differences between 2 groups were compared by usingan unpaired 2-tailed Student t test, while differences between≥3 groups were compared by using analysis of variance withthe Bonferroni posttest. Data were considered statistically signif-icant at a P value of < .05.

90 • JID 2014:210 (1 July) • Yang et al


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RESULTS

P. aeruginosa Infection Induces miR-155 Expression in Humanand Mouse CorneasTo determine whether there is a clinical relationship betweenmiR-155 and P. aeruginosa keratitis, we examined miR-155 ex-pression in P. aeruginosa–infected human corneas (clinicalspecimens) and normal uninfected corneas from healthy do-nors. The results showed that miR-155 expression was signifi-cantly upregulated in human corneas after P. aeruginosainfection (Figure 1A). The expression levels of miR-155 inP. aeruginosa–infected corneas were approximately 25-foldhigher 1–6 days after infection, 40-fold higher 7–13 days afterinfection, and 120-fold higher 14–30 days after infection, com-pared with levels in uninfected corneas from healthy donors(P < .001 for all comparisons), suggesting that miR-155 is clini-cally relevant to P. aeruginosa keratitis. We further tested miR-155expression in a well-established murine model of P. aeruginosakeratitis. miR-155 expression was significantly enhanced in B6mouse corneas 1, 3, and 5 days after infection (P < .001 for

all comparisons; Figure 1B), which is consistent with the clinicaldata.

miR-155 Is Mainly Expressed in Macrophages but NotNeutrophilsTo further determine the cell source of P. aeruginosa–inducedmiR-155 expression, we next investigated the miR-155 expres-sion in primary PMNs and macrophages isolated from mousebone marrow before and after P. aeruginosa infection. Polymer-ase chain reaction (PCR) data showed that miR-155 expressionlevels in bone marrow–derived PMNs were much lower thanthose in macrophages, with or without P. aeruginosa infection(Figure 1C). Similarly, P. aeruginosa–challenged human mono-cyte–derived macrophages (MDMs) displayed much highermiR-155 expression than human PMNs (Figure 1D). To furtherconfirm the cell source of miR-155 in vivo, we sorted PMNs andmacrophages from P. aeruginosa–infected mouse corneas, usingflow cytometry (Figure 1E ). The expression of miR-155 in sort-ed PMNs (Gr-1 positive) or macrophages (F4/80 positive) wasdetected using single-cell PCR. Our data showed that the

Figure 1. Pseudomonas aeruginosa infection induces miR-155 (microRNA-155) expression in both human and mouse corneas and macrophages. A, miR-155 expression in normal and P. aeruginosa–infected human corneas 1–6 days after infection, 7–13 days after infection, and 14–30 days after infection.Data are mean ± standard error of the mean (SEM) with 8 patients/group. B, miR-155 expression in normal and P. aeruginosa–infected B6 corneas 1, 3, and5 days after infection. Data are mean ± SEM with 10 mice/time points and represent 3 individual experiments. C, D, G, and H, miR-155 expression in murinepolymorphonuclear neutrophils (PMNs) versus bone marrow–derived macrophages, human PMNs versus human monocyte–derived macrophages, murineperitoneal macrophages, and RAW264.7 cells after P. aeruginosa challenge at a multiplicity of infection of 1 for the indicated time points. E, Fluorescence-activated cell sorting of corneal cell suspension 3 days after infection, following Gr-1 and F4/80 staining. F, miR-155 expression in sorted PMNs and mac-rophages. Data are mean ± SEM and represent 3 individual experiments. **P < .01; ***P < .001.



expression of miR-155 in macrophages was approximately 32-fold higher than that in PMNs (Figure 1F ). These results indi-cate that macrophages are the major inflammatory cell source ofmiR-155 in P. aeruginosa keratitis. We further challenged mu-rine peritoneal macrophages (Figure 1G) and macrophage-likeRAW264.7 cells (Figure 1H) with P. aeruginosa at indicatedtimes. Real-time PCR data showed that miR-155 expressionwas dramatically enhanced in a time-dependent manner inboth macrophages cell types.

miR-155 Contributes to Corneal Susceptibility to P. aeruginosaInfectionTo investigate whether miR-155 participates in P. aeruginosakeratitis, miR-155 KO and WT B6 mice were infected with P.aeruginosa. miR-155 KO mice displayed a highly resistant phe-notype against P. aeruginosa ocular infection, compared withWT B6 mice, as shown by the decreased clinical score amongmiR-155 KO mice 3 and 5 days after infection (P < .05 andP < .001, respectively; Figure 2A). Representative photographsof corneas from miR-155 KO and WT mice 5 days after infec-tion are provided in Figure 2C and 2B, respectively. By 5 daysafter infection, miR-155 KO mice displayed less severe cornealdisease (clinical score, +2/+3), whereas corneas from most WTmice were perforated (clinical score, +4). Together, these datasuggest that miR-155 promotes host susceptibility to P. aerugi-nosa infection.

miR-155 Enhances Bacterial Burden in P. aeruginosa KeratitisSince in vivo studies indicated that deficiency of miR-155 pro-moted host resistance to P. aeruginosa corneal infection, wenext assessed the effect of miR-155 on the bacterial componentof disease pathogenesis, using a plate count assay. A reducedbacterial load was detected in the infected corneas of miR-155KO mice, compared with WT mice, 5 days after infection(P < .01), whereas no difference between these 2 groups was de-tected 1 day after infection (Figure 2D). Moreover, 5 days afterinfection, messenger RNA levels of inducible nitric oxide syn-thase (iNOS) were elevated in miR-155 KO mice (P < .001),whereas levels of messenger RNA expression of the followingwere unchanged between the miR-155 KO and WT groups:NOX2 (NADPH oxidase), an enzyme for reactive oxygen spe-cies generation; CRAMP (cathelicidin-related antimicrobialpeptide); and mBD (murine beta defensin) 2 and 3 (Figure 2E).

miR-155 Inhibits Macrophage-Mediated Phagocytosis ofP. aeruginosaWe further explored the mechanism of miR-155–mediated im-mune regulation by using murine bone marrow–derived macro-phages (BMDMs) and macrophage-like RAW264.7 cells. Platecount data showed that the number of bacteria engulfed byBMDMs within 1 hour after challenge was enhanced in miR-155 KO mice, compared with WT mice (Figure 3A). Moreover,phagocytosis of P. aeruginosa in miR-155–overexpressed versus

Figure 2. miR-155 (microRNA-155) knockout (KO) mice are resistant toPseudomonas aeruginosa corneal infection. miR-155 KO and wild-type B6mice were infected with P. aeruginosa. A, Clinical score was recorded foreach mouse 1, 3, and 5 days after infection. Representative photographs of in-fected eyes in wild-type (B) versus miR-155 KO mice (C) were taken 5 days afterinfection. D, Bacterial load in the infected cornea was examined by plate countin wild-type versus miR-155 KO mice 1 and 5 days after infection. E, MessengerRNA (mRNA) expression levels of inducible nitric oxide synthase (iNOS), NOX2(NADPH oxidase), CRAMP (cathelicidin-related antimicrobial peptide), mBD2(murine beta defensin 2), and mBD3 in the infected corneas of miR-155 KOversus wild-type mice were examined at 5 days after infection by real-timepolymerase chain reaction. Data are mean ± standard error of the mean andrepresent 3 individual experiments, each with 5 animals per group per timeper assay. *P < .05; **P < .01; ***P < .001. Abbreviation: NS, not significant.



control-treated RAW264.7 cells was assessed using plate count,immunostaining, and flow cytometry. Plate count data demon-strated that phagocytosis of P. aeruginosa was significantlyreduced after miR-155 overexpression, compared with con-trol treatment (P < .001; Figure 3B). Immunostaining data(Figure 3C) showed less colocalization of FilmTracer GreenBiofilm–labeled P. aeruginosa with Texas red–labeled cellsafter miR-155 mimic overexpression, compared with control

treatment, indicating that the number of P. aeruginosa phagocy-tosed by RAW264.7 cells was decreased by miR-155 overexpres-sion. Results of flow cytometry (Figure 3D) also indicated thatmiR-155 suppressed macrophage-mediated P. aeruginosaphagocytosis by 75%, as calculated by the mean fluorescenceintensity (P < .01; Figure 3E ). These data together provide evi-dence that miR-155 inhibits macrophage-mediated phagocyto-sis of P. aeruginosa.

Figure 3. miR-155 (microRNA-155) inhibits macrophage-mediated phagocytosis of Pseudomonas aeruginosa. A, Phagocytosis of P. aeruginosa in bonemarrow–derived macrophages isolated from miR-155 knockout (KO) mice versus wild-type B6 mice was examined by plate count. B–E, RAW264.7 cells weretransfected with miR-155 or control mimic for 24 hours and then infected with P. aeruginosa. Uptake of P. aeruginosawas detected by a phagocytosis assayusing plate count (B), immunofluorescence (C; P. aeruginosa is indicated by the small light colored ovals, and RAW264.7 cells are indicated by the darkershading) and flow cytometry (D, mean fluorescence intensity (MFI) was calculated and is shown in E ), respectively. Data are mean ± standard error of themean and represent 3 individual experiments. **P < .01. Abbreviations: FTGB, FilmTracer Green Biofilm; NS, not significant.



miR-155 Suppresses Macrophage-Mediated Bacterial Killing ofP. aeruginosaNext, we investigated the role of miR-155 in intracellular bacte-rial killing by means of a plate count assay. BMDMs isolatedfrom miR-155 KO mice displayed higher capability of bacterialkilling than WT mice (P < .01; Figure 4A). On the other hand,miR-155 overexpression in RAW264.7 cells significantly re-duced the killing of engulfed bacteria (P < .001; Figure 4B).Moreover, NO production was significantly reduced by miR-155 overexpression 12, 24, and 48 hours after P. aeruginosachallenge (P < .001 for all comparisons; Figure 4C), and iNOSexpression was decreased in miR-155–overexpressed cells,

compared with control RAW264.7 cells, 6, 12, 24, and 48 hoursafter infection (P < .01, P < .05, P < .01, and P < .01, respectively;Figure 4D). For reactive oxygen species production, no changewas detected between the 2 groups (Figure 4E). These data indi-cate that miR-155 inhibits macrophage-mediated bacterial killingof P. aeruginosa by reducing NO production.

miR-155 Suppresses Phagocytosis and Intracellular Killing byTargeting RhebSince the function of microRNA is largely dependent on itstargets, we next searched for the specific targets of miR-155–mediated bacterial clearance. We first tested the role of

Figure 4. miR-155 (microRNA-155) inhibits macrophage-mediated bacterial killing of Pseudomonas aeruginosa by reducing NO production. A, Killing ofP. aeruginosa in bone marrow–derived macrophages isolated from miR-155 knockout (KO) mice, compared with wild-type B6 mice was examined by platecount. B–E, RAW264.7 cells were transfected with miR-155 or control mimic for 24 hours and then infected with P. aeruginosa. Intracellular killing ofP. aeruginosa was detected by plate count (B). NO production (C) and inducible nitric oxide synthase (iNOS) expression levels (D) were tested by Griessreaction and real-time polymerase chain reaction, respectively. Reactive oxygen species generation was detected by flow cytometry (E ). Data aremean ± standard error of the mean (n = 4) and represent 3 individual experiments. *P < .05; **P < .01; ***P < .001. Abbreviations: mRNA, messengerRNA; NS, not significant.



SHIP1, a well-established target of miR-155, on P. aeruginosaclearance. Western blot data showed that overexpression ofmiR-155 decreased the expression of SHIP1 (SupplementaryFigure 1A). However, knockdown of SHIP1 (SupplementaryFigure 1B) had no effect on macrophage-mediated phagocytosis(Supplementary Figure 1C and 1D) or bacterial killing ofP. aeruginosa (Supplementary Figure 1E ), indicating thatSHIP1 was not involved in miR-155–mediated inhibition ofP. aeruginosa clearance. Our recent study showed that miR-155

posttranscriptionally suppresses Rheb by targeting its 3′ un-translated region [18].Western blot data showed that Rheb pro-tein levels were decreased after miR-155 overexpression(Figure 5A) and increased after miR-155 inhibition (Figure 5B)in RAW264.7 cells, with or without P. aeruginosa infection.Next, we examined the effect of Rheb knockdown on phagocy-tosis and bacterial killing. The knockdown efficacy was con-firmed by Western blot (Figure 5C). Both flow cytometry(Figure 5D) and bacteria plate count (Figure 5E) data showed

Figure 5. miR-155 (microRNA-155) suppresses macrophage-mediated phagocytosis and killing of Pseudomonas aeruginosa by targeting Rheb (Ras ho-molog enriched in brain). A and B, Protein levels of Rheb were tested by Western blot in RAW264.7 cells transfected with miR-155 mimic, compared withcontrol mimic, or with LNA–anti-miR-155, compared with control LNA, before and after P. aeruginosa infection. Band intensity was quantitated andnormalized to the β-actin. C–G, RAW264.7 cells were transfected with small interfering RNAs (siRNAs) targeting Rheb for 48 hours, followed byP. aeruginosa challenge. H–K, RAW264.7 cells stably expressing Rheb (RAW-Rheb) or control vector (RAW-control) were transfected with miR-155 or controlmimic for 24 hours, followed by P. aeruginosa challenge. Rheb expression was tested by Western blot (C and I). Phagocytosis was detected by flow cy-tometry (D) and plate count assay (E and J). Intracellular killing (H and L) of P. aeruginosawas detected by plate count assay. NO production (F ) and induciblenitric oxide synthase (iNOS) expression levels (G and K ) were tested by Griess reaction and real-time polymerase chain reaction, respectively. Data aremean ± standard error of the mean (n = 4) and represent 3 individual experiments. *P < .05; **P < .01; ***P < .001. Abbreviation: NS, not significant.










that knockdown of Rheb inhibited macrophage-mediatedphagocytosis of P. aeruginosa. Moreover, iNOS expres-sion and NO production were reduced after silencing Rheb inP. aeruginosa–challenged RAW264.7 cells (Figure 5F and 5G).Plate count data showed that bacterial killing was reducedafter knockdown of Rheb (Figure 5H). To further determinewhether Rheb is the major target of miR-155 in bacterial clear-ance, we examined the inhibitory effect of miR-155 on phago-cytosis and bacterial killing in RAW264.7 cells with ectopicexpression of Rheb (Figure 5I). Results of a phagocytosisassay based on bacterial plate count indicated that overexpres-sion of miR-155 inhibited macrophage-mediated bacterial up-take in control RAW264.7 cells, but this inhibitory effect wasblocked in RAW264.7 cells stably expressing Rheb (RAW-Rheb; Figure 5J). In addition, overexpression of miR-155 re-duced iNOS expression and macrophage-mediated killing ofP. aeruginosa, whereas this ability was blocked after overexpres-sion of Rheb (Figure 5K and 5L). These data together indicatethat miR-155 modulates bacterial clearance by targeting Rheb.

DISCUSSION

microRNAs have emerged as novel posttranslational regulatorsin various physiological and pathological events, such as cellproliferation, differentiation, apoptosis, and cytokine produc-tion [15, 19–21]. Nonetheless, the molecular basis involved inthe microRNA-mediated ocular immune regulation remainslargely unclear. Here we report an unexplored role of miR-155 in regulating bacterial elimination in P. aeruginosa keratitisby targeting Rheb, which may provide a better understanding ofthe host antibacterial response.

First, our data show that miR-155 expression is dramaticallyincreased in both human and mouse corneas after P. aeruginosainfection. The induction of miR-155 is closely associated with in-fection duration, indicating the potential participation of miR-155 in the progression of P. aeruginosa keratitis. Moreover, ourin vivo studies show that miR-155 KO mice displayed more resis-tance to P. aeruginosa keratitis, compared with WT B6 mice, in-dicating that the presence of miR-155 contributes to the cornea’ssusceptibility. Accumulating evidence demonstrates that duringP. aeruginosa keratitis, bacterial virulence is the major factor lead-ing to the disease pathogenesis. Lower bacterial load was observedin corneas from miR-155 KO mice, compared with those fromWT mice, 5 days after infection, suggesting that miR-155 playsan inhibitory role in bacterial clearance.

The innate immunity is the first line of host defense againstinvading pathogens. During P. aeruginosa keratitis, a largeamount of phagocytes, including PMNs and macrophages, arerapidly recruited to the infection site and kill bacteria. It is report-ed that miR-155 expression is upregulated in G-CSF–mobilizedCD34-positive mononuclear and neutrophil phagocyte precur-sors [22]. Our in vitro and in vivo studies indicate that during

P. aeruginosa infection, both constitutive and inducible miR-155 expression levels are much higher in macrophages than inneutrophils, suggesting that macrophages are the major cellsource of P. aeruginosa–triggered miR-155 induction.

Macrophages play a critical role in determining the outcomeof P. aeruginosa keratitis. Studies have demonstrated that deple-tion of macrophages in susceptible B6 mice decreased bacterialclearance and increased the severity of P. aeruginosa keratitis[1]. Since miR-155 is mainly expressed in macrophages, its invivo microbicidal effect is probably dependent on modulatingmacrophages activity. Macrophages express a series of phago-cytic receptors that help to facilitate the internalization of invad-ing pathogens. It was reported that miR-155 expression wasdecreased in syngeneic bone marrow transplant alveolar macro-phages and that inhibition of miR-155 in alveolar macrophagesincreased phagocytosis of Staphylococcus aureus [23]. Our datademonstrate that miR-155 suppresses phagocytosis of P. aerugi-nosa in macrophages, providing further evidence regarding therole of miR-155 in bacterial clearance. Once bacteria are en-gulfed by macrophages, they are subjected to intracellular killingvia oxygen-dependent systems, such as those involving reactiveoxygen species and reactive nitrogen species, and oxygen-inde-pendent systems, such as those involving antimicrobial pep-tides. Hazlett et al have demonstrated that during P.aeruginosa keratitis, iNOS-derived NO is required for bacterialkilling, and the macrophage is the major cell source of NO [24].While Wu et al found that mBD2 (expressed on macrophages,PMNs, and fibroblasts) and mBD3 (expressed on PMNs) are re-quired in host resistance against P. aeruginosa corneal infectionby enhancing bacterial killing and reducing inflammatory re-sponse [25, 26]. Kumar R et al reported that inhibition ofmiR-155 reduced the survival of Mycobacterium tuberculosisin macrophages, although the underlying mechanism of en-hanced bacterial killing was not clear [27]. Our in vivo and invitro studies demonstrate that miR-155 suppresses bacterialclearance and NO production but not reactive oxygen speciesor antimicrobial peptide production, indicating that miR-155reduces intracellular killing of P. aeruginosa by inhibiting NOproduction. In addition, there are 2 types of macrophage activa-tion: classical (M1) activation, which promotes bacterial killingand tissue damage, and alternative (M2) activation, which playsa critical role in tissue repair. Our study demonstrates that in theP. aeruginosa–infected macrophages, miR-155 suppresses theexpression of iNOS, a well-known marker of M1 polarization,implying that miR-155 may inhibit bacterial clearance by inhib-iting an M1-like macrophage activation. It is worthwhile tomention that several studies reported that human macrophagesdo not produce NO because of lacking exogenous tetrahydro-biopterin, an essential cofactor indispensible for iNOS activity[28–32]. Our data demonstrate that in human MDMs, P. aeru-ginosa infection does not induce NO production, but overex-pression of miR-155 reduces the expression of NOX2, a key



synthase for reactive oxygen species generation (data notshown). These data together indicated that miR-155 may usedifferent antimicrobial mechanisms in human and murinemacrophages.

Studies have demonstrated that miR-155 executes differentactivities in various physical, pathological, and experimentalconditions by repressing distinct targets, such as SOCS1 [33],C/EBP β (CCAAT/enhancer binding protein β) [34], andSHIP1 [35]. It is also reported that miR-155 decreased NO pro-duction in human umbilical vein endothelial cells and acetyl-choline-induced endothelium-dependent vasorelaxation bytargeting endothelial NOS [36]. However, most of the reportedmiR-155 targets are linked to inflammatory regulation, andnothing is known regarding the specific target involved in mod-ulating bacterial phagocytosis and killing.

Our previous study identified that miR-155 targets to the 3′untranslated region of Rheb and suppresses Rheb expression ata posttranscriptional level [18]. In the present study, we foundthat regardless of the presence of P. aeruginosa infection, over-expression of miR-155 decreased, whereas inhibition of miR-155increased the protein levels of Rheb in macrophages. Further-more, miR-155–mediated inhibition of bacterial phagocytosisand killing are blocked in RAW264.7 cells stably expressingRheb, indicating that Rheb is the major target of miR-155 inmodulating macrophage-mediated bacterial elimination. It isreported that Rheb can directly interact with mTOR (mamma-lian target of rapamycin) [37] and increase mTOR activity [38].A recent study using the same murine model of P. aeruginosakeratitis has demonstrated that inhibition of mTOR in B6 miceby treatment with rapamycin increased the clinical score andbacterial load but diminished PMN bactericidal activity in re-sponse to P. aeruginosa corneal infection [39]. Therefore, wespeculate that miR-155 enhances corneal susceptibility toP. aeruginosa keratitis by targeting Rheb and suppressing bacte-rial clearance.

We also tested the role of SHIP1, a well-established target ofmiR-155 [13, 27, 35] that has been reported to participate inFcR-mediated phagocytosis of immunoglobulin G (IgG)–opsonized erythrocytes [40]. Overexpression of miR-155 inhib-ited SHIP1 protein expression in macrophages with or withoutP. aeruginosa challenge. However, in our P. aeruginosa infectionmodel, knockdown of SHIP1 expression had little effect onphagocytosis and killing of P. aeruginosa. This discrepancymay be attributed to different phagocytosis mechanisms ofIgG-opsonized erythrocytes and P. aeruginosa.

Collectively, our study demonstrates that miR-155 reducesmacrophage-mediated bacterial clearance by targeting Rheband thus contributes to corneal susceptibility to P. aeruginosakeratitis. To our knowledge, it is the first study to explore therole of miR-155 in P. aeruginosa killing, which may illustratesome implications for the design of microRNA-based treatmentof bacterial keratitis and other infectious diseases.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseasesonline (http://jid.oxfordjournals.org/). Supplementary materials consist ofdata provided by the author that are published to benefit the reader. Theposted materials are not copyedited. The contents of all supplementarydata are the sole responsibility of the authors. Questions or messages regardingerrors should be addressed to the author.

Notes

Acknowledgments. We thank Dr Linda Hazlett and Dr Harley Tse fromWayne State University School of Medicine, for their useful comments andlanguage editing.Financial support. This work was supported by the National Natural

Science Foundation of China (grants U0832006 and 81261160323 [toX. H.], 31200662 and 31370868 [to M. W.], and 81172811 [to M. L.], theState Key Laboratory of Ophthalmology (Zhongshan Ophthalmic Center;Open Project Grant to X. H. and J. Y.), the Guangdong Innovative ResearchTeam Program (2009010058 [to X. H.] and 2011Y035 [to M. W.]), the Doc-toral Program of Higher Education of China (Specialized Research Funds20100171110047 [to X. H.] and 20120171120064 [to M. W.]), the Guang-dong Natural Science Foundation (10251008901000013 [to X. H.] andS2012040006680 [to M. W.]), the 111 Project (B13037 to M. W.), Guang-dong Province Universities and Colleges (Pearl River Scholar FundedScheme 2009 to X. H.), and the National Science and Technology Key Pro-jects for Major Infectious Diseases (2013ZX10003001 to X. H.).Potential conflicts of interest. All authors: No reported conflicts.All authors have submitted the ICMJE Form for Disclosure of Potential

Conflicts of Interest. Conflicts that the editors consider relevant to thecontent of the manuscript have been disclosed.

References

1. Hazlett LD. Corneal response to Pseudomonas aeruginosa infection.Prog Retin Eye Res 2004; 23:1–30.

2. Iglewski BH, Liu PV, Kabat D. Mechanism of action of Pseudomonasaeruginosa exotoxin Aiadenosine diphosphate-ribosylation of mamma-lian elongation factor 2 in vitro and in vivo. Infect Immun 1977; 15:138–44.

3. Schultz CL, Morck DW, McKay SG, Olson ME, Buret A. Lipopolysac-charide induced acute red eye and corneal ulcers. Exp Eye Res 1997;64:3–9.

4. Feterl M, Govan BL, Ketheesan N. The effect of different Burkholderiapseudomallei isolates of varying levels of virulence on toll-like-receptorexpression. Trans R Soc Trop Med Hyg 2008; 102(Suppl 1):S82–8.

5. Cowell BA, Willcox MD, Hobden JA, Schneider RP, Tout S, Hazlett LD.An ocular strain of Pseudomonas aeruginosa is inflammatory but notvirulent in the scarified mouse model. Exp Eye Res 1998; 67:347–56.

6. Hazlett LD, Zelt R, Cramer C, Berk RS. Pseudomonas aeruginosa in-duced ocular infection. A histological comparison of two bacterialstrains of different virulence. Ophthalmic Res 1985; 17:289–96.

7. Hazlett LD, McClellan S, Kwon B, Barrett R. Increased severity of Pseu-domonas aeruginosa corneal infection in strains of mice designated asTh1 versus Th2 responsive. Invest Ophthalmol Vis Sci 2000; 41:805–10.

8. Mulik S, Xu J, Reddy PB, et al. Role of miR-132 in angiogenesis afterocular infection with herpes simplex virus. Am J Pathol 2012; 181:525–34.

9. Kandalam MM, Beta M, Maheswari UK, Swaminathan S, Krishnaku-mar S. Oncogenic microRNA 17–92 cluster is regulated by epithelialcell adhesion molecule and could be a potential therapeutic target in ret-inoblastoma. Mol Vis 2012; 18:2279–87.

10. Escobar T, Yu CR, Muljo SA, Egwuagu CE. STAT3 activates miR-155 inTh17 cells and acts in concert to promote experimental autoimmuneuveitis. Invest Ophthalmol Vis Sci 2013; 54:4017–25.




http://jid.oxfordjournals.org/

http://jid.oxfordjournals.org/

11. Yao R, Ma YL, LiangW, et al. MicroRNA-155 modulates Treg and Th17cells differentiation and Th17 cell function by targeting SOCS1. PLoSOne 2012; 7:e46082.

12. Suzuki HI, Arase M, Matsuyama H, et al. MCPIP1 ribonuclease antag-onizes dicer and terminates microRNA biogenesis through precursormicroRNA degradation. Mol Cell 2011; 44:424–36.

13. Cremer TJ, Ravneberg DH, Clay CD, et al. MiR-155 inductionby F. novicida but not the virulent F. tularensis results in SHIP down-regulation and enhanced pro-inflammatory cytokine response. PLoSOne 2009; 4:e8508.

14. Ceppi M, Pereira PM, Dunand-Sauthier I, et al. MicroRNA-155modulates the interleukin-1 signaling pathway in activated humanmonocyte-derived dendritic cells. Proc Natl Acad Sci U S A 2009; 106:2735–40.

15. He M, Xu Z, Ding T, Kuang DM, Zheng L. MicroRNA-155 regulatesinflammatory cytokine production in tumor-associated macrophagesvia targeting C/EBPbeta. Cell Mol Immunol 2009; 6:343–52.

16. Wu M, Peng A, Sun M, et al. TREM-1 amplifies corneal inflammationafter Pseudomonas aeruginosa infection by modulating Toll-like recep-tor signaling and Th1/Th2-type immune responses. Infect Immun2011; 79:2709–16.

17. Chen K, Yin L, Nie X, et al. beta-Catenin promotes host resistanceagainst Pseudomonas aeruginosa keratitis. J Infect 2013; 67:584–94.

18. Wang J, Yang K, Zhou L, et al. MicroRNA-155 promotes autophagy toeliminate intracellular mycobacteria by targeting Rheb. PLoS Pathog2013; 9:e1003697.

19. Xiao C, Rajewsky K. MicroRNA control in the immune system: basicprinciples. Cell 2009; 136:26–36.

20. Wu S, He L, Li Y, et al. miR-146a facilitates replication of dengue virusby dampening interferon induction by targeting TRAF6. J Infect 2013;67:329–41.

21. Niu Y, Mo D, Qin L, et al. Lipopolysaccharide-induced miR-1224 neg-atively regulates tumour necrosis factor-alpha gene expression by mod-ulating Sp1. Immunology 2011; 133:8–20.

22. Donahue RE, Jin P, Bonifacino AC, et al. Plerixafor (AMD3100) andgranulocyte colony-stimulating factor (G-CSF) mobilize differentCD34+ cell populations based on global gene and microRNA expres-sion signatures. Blood 2009; 114:2530–41.

23. Domingo-Gonzalez R, Katz S, Serezani CH, Moore TA, Levine AM,Moore BB. Prostaglandin E2-induced changes in alveolar macrophagescavenger receptor profiles differentially alter phagocytosis of Pseudo-monas aeruginosa and Staphylococcus aureus post-bone marrow trans-plant. J Immunol 2013; 190:5809–17.

24. Hazlett LD, McClellan S, Goshgarian C, Huang X, Thakur A, Barrett R.The role of nitric oxide in resistance to P. aeruginosa ocular infection.Ocul Immunol Inflamm 2005; 13:279–88.

25. Wu M, McClellan SA, Barrett RP, Hazlett LD. Beta-defensin-2 pro-motes resistance against infection with P. aeruginosa. J Immunol2009; 182:1609–16.

26. Wu M, McClellan SA, Barrett RP, Zhang Y, Hazlett LD. Beta-defensins2 and 3 together promote resistance to Pseudomonas aeruginosa kerati-tis. J Immunol 2009; 183:8054–60.

27. Kumar R, Halder P, Sahu SK, et al. Identification of a novel role ofESAT-6-dependent miR-155 induction during infection of macrophag-es with Mycobacterium tuberculosis. Cell Microbiol 2012; 14:1620–31.

28. Murray HW, Teitelbaum RF. L-arginine-dependent reactive nitrogenintermediates and the antimicrobial effect of activated human mononu-clear phagocytes. J Infect Dis 1992; 165:513–7.

29. Schneemann M, Schoedon G, Hofer S, Blau N, Guerrero L, Schaffner A.Nitric oxide synthase is not a constituent of the antimicrobial armatureof human mononuclear phagocytes. J Infect Dis 1993; 167:1358–63.

30. Weinberg JB, Misukonis MA, Shami PJ, et al. Human mononuclearphagocyte inducible nitric oxide synthase (iNOS): analysis of iNOSmRNA, iNOS protein, biopterin, and nitric oxide production by bloodmonocytes and peritoneal macrophages. Blood 1995; 86:1184–95.

31. Denis M. Human monocytes/macrophages: NO or no NO? J LeukocBiol 1994; 55:682–4.

32. Albina JE. On the expression of nitric oxide synthase by human macro-phages. Why no NO? J Leukoc Biol 1995; 58:643–9.

33. Wang P, Hou J, Lin L, et al. Inducible microRNA-155 feedback pro-motes type I IFN signaling in antiviral innate immunity by targetingsuppressor of cytokine signaling 1. J Immunol 2010; 185:6226–33.

34. O’Connell RM, Rao DS, Chaudhuri AA, et al. Sustained expression ofmicroRNA-155 in hematopoietic stem cells causes a myeloproliferativedisorder. J Exp Med 2008; 205:585–94.

35. O’Connell RM, Chaudhuri AA, Rao DS, Baltimore D. Inositol phospha-tase SHIP1 is a primary target of miR-155. Proc Natl Acad Sci U S A2009; 106:7113–8.

36. Sun HX, Zeng DY, Li RT, et al. Essential role of microRNA-155 in reg-ulating endothelium-dependent vasorelaxation by targeting endothelialnitric oxide synthase. Hypertension 2012; 60:1407–14.

37. Sanjuan MA, Milasta S, Green DR. Toll-like receptor signaling in thelysosomal pathways. Immunol Rev 2009; 227:203–20.

38. Garami A, Zwartkruis FJ, Nobukuni T, et al. Insulin activation of Rheb,a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2.Mol Cell 2003; 11:1457–66.

39. Foldenauer ME, McClellan SA, Berger EA, Hazlett LD. Mammalian tar-get of rapamycin regulates IL-10 and resistance to Pseudomonas aeru-ginosa corneal infection. J Immunol 2013; 190:5649–58.

40. Kamen LA, Levinsohn J, Swanson JA. Differential association of phos-phatidylinositol 3-kinase, SHIP-1, and PTEN with forming phago-somes. Mol Biol Cell 2007; 18:2463–72.



mir-155 suppresses bacterial clearance in pseudomonas

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