MAJOR ARTICLE

miR-155 Suppresses Bacterial Clearance in Pseudomonas aeruginosa–Induced Keratitis by Targeting 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 1

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Department of Immunology, Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 2Key Laboratory of Tropical 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 Pseudomonas aeruginosa–induced keratitis, one of the most common sight-threatening ocular diseases. We found that miR-155 expression was enhanced in human and mouse corneas after P. aeruginosa infection and was mainly expressed in macrophages but not neutrophils. In vivo studies demonstrated that miR-155 knockout mice displayed more resistance to P. aeruginosa keratitis, with a higher inducible nitric oxide synthase level and a lower bacterial burden. More importantly, in vitro data indicated that miR-155 suppressed the macrophage-mediated bacterial phagocytosis and intracellular killing of P. aeruginosa by targeting Rheb (Ras homolog enriched in brain). To the best of our knowledge, this is the first study to explore the role of miR-155 in bacterial keratitis, which may provide a promising target for clinical treatment of P. aeruginosa keratitis and other infectious diseases. Keywords. Rheb.

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

Pseudomonas aeruginosa is one of the bacterial species most commonly isolated from contact-lens users with corneal infection (keratitis) [1]. P. aeruginosa– induced keratitis is a suppurative ocular infectious disease that progresses rapidly and often leads to inflammatory epithelial edema, stromal infiltration, tissue destruction, and corneal ulceration and sometimes leads to vision loss [1]. Invading P. aeruginosa replicates within the host body and produces a variety of virulence factors, such as exotoxin A [2], endotoxin lipopolysaccharide [3], and exoenzyme ExoU [4]. These virulence factors not only induce the death of host cells by destroying the

Received 15 September 2013; accepted 31 December 2013; electronically published 7 January 2014. a K. 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 Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: [email protected]. DOI: 10.1093/infdis/jiu002

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 neutrophils (PMNs) and macrophages are quickly recruited to the infection site to engulf invading microorganisms [1]. Activated macrophages and PMNs produce a large amount of reactive oxygen species and reactive nitrogen species to kill the engulfed bacteria, thereby controlling the bacterial burden in the infected cornea. Experimental P. aeruginosa challenge usually induces corneal perforation in susceptible B6 mice (T-helper type 1 responders) at 5 days after infection, but disease is much less severe in resistant BALB/c mice (T-helper type 2 responders) [7], indicating the importance of immune regulation in determining the disease outcome. Recently, several microRNAs have been implicated as critical regulators in ocular diseases, such as miR-132 in herpes simplex virus–induced keratitis and corneal neovascularization [8], miR17-92 cluster in retinoblastoma cell proliferation and invasion [9], and miR-155 in

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Patients and Tissue Specimens

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

Eight-week-old female wild-type (WT) or miR-155 knockout (KO) C57BL/6 (B6) mice were from Jackson Laboratory (Bar Harbor, ME). Infection of mouse corneas and scoring of corneal disease were performed following the protocol reported elsewhere [16, 17], as described in the Supplementary Materials. Plate Count to Assess Phagocytosis and Intracellular Killing

For studies using clinical human materials, written informed consent from participating patients and approval from the Research Ethics Committee of Sun Yat-sen University (Guangzhou, China) were obtained. For animal studies, all of the experiments were performed in accordance with the guidelines provided in the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Scientific Investigation Board of Sun Yat-sen University.

Phagocytosis and intracellular killing were assessed by plate count, as described by others [4]. Cells were challenged with P. aeruginosa at a multiplicity of infection of 25. The numbers of internalized and killed bacteria were assessed after 1 or 2 hours of incubation. After 1 hour, gentamicin was added to the medium at 300 µg/mL for 30 minutes to kill extracellular bacteria. Cells were then lysed, and bacterial colony-forming units (CFU) were determined by a plate count assay. The phagocytosis efficiency was calculated on the basis of CFU data obtained 1 hour after infection and was normalized to the control group. A second series of internalization assays was run in parallel to determine the number of viable bacteria following 2 hours of incubation. For this, after the same treatment to remove extracellular bacteria, cells were incubated for another 1 hour and then lysed for analysis of intracellular bacterial CFU. The killing efficiency was calculated as [CFU (1 hour) – CFU (2 hour)]/CFU (1 hour) and normalized to the control group.

Reagents

Statistical Analysis

P. aeruginosa strain 19660 was from ATCC. Control and miR155 mimics were from Applied Biosystems (Life Technologies, Grand Island, NY). LNA control and LNA miR-155 inhibitors were from Exiqon (Vedbaek, Denmark). Anti-Rheb antibody was from Abcam (New Territories, Hong Kong). Anti-SHIP1 antibody was from Cell Signaling Technology (Beverly, MA). TaqMan microRNA assay kits were from Applied Biosystems.

The differences in clinical score between 2 groups at each time point were tested by the Mann–Whitney U test. For other experiments, differences between 2 groups were compared by using an unpaired 2-tailed Student t test, while differences between ≥3 groups were compared by using analysis of variance with the Bonferroni posttest. Data were considered statistically significant at a P value of < .05.

METHODS Ethics Statements

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experimental autoimmune uveitis and expansion of pathogenic T-helper type 17 cells [10]. However, nothing is known regarding the role of microRNAs in modulating bacterial keratitis. As one of the most studied microRNAs in the field of immune regulation, miR-155 executes multiple functions by targeting distinct messenger RNAs. Studies have demonstrated that miR-155 enhances T-regulatory cell and T-helper type 17 cell differentiation and T-helper type 17 cell function by targeting SOCS1 (suppressor of cytokine signaling 1) [11], and attenuates T-helper type 2 cell responses by targeting c-Maf, a potent transactivator of the interleukin 4 promoter [12]. It is also reported that miR-155 enhanced proinflammatory cytokine response to Francisella novicida infection by targeting SHIP-1 (Src homology-2 domain-containing inositol 5-phosphatase 1) [13] but functions as a negative regulator of inflammation in endotoxin lipopolysaccharide–challenged human dendritic cells, by targeting TAB2 (transforming growth factor β–activated kinase 1/MAP3K7 binding protein 2) [14, 15]. Nonetheless, whether miR-155 is involved in P. aeruginosa clearance and its downstream target remain unclear. In the present study, we investigated the potential role of miR-155 in P. aeruginosa keratitis. Our study demonstrated that miR-155 expression was significantly induced after P. aeruginosa infection in vivo and in vitro, and contributed to corneal susceptibility by suppressing bacterial eradication. More importantly, we identified Rheb (Ras homolog enriched in brain) as a novel functional target of miR-155 in attenuating macrophage-mediated bacterial phagocytosis and intracellular killing. These data may provide a promising therapeutic target for the treatment of bacterial keratitis and other infectious diseases.

RESULTS P. aeruginosa Infection Induces miR-155 Expression in Human and Mouse Corneas

To determine whether there is a clinical relationship between miR-155 and P. aeruginosa keratitis, we examined miR-155 expression in P. aeruginosa–infected human corneas (clinical specimens) and normal uninfected corneas from healthy donors. The results showed that miR-155 expression was significantly upregulated in human corneas after P. aeruginosa infection (Figure 1A). The expression levels of miR-155 in P. aeruginosa–infected corneas were approximately 25-fold higher 1–6 days after infection, 40-fold higher 7–13 days after infection, and 120-fold higher 14–30 days after infection, compared with levels in uninfected corneas from healthy donors (P < .001 for all comparisons), suggesting that miR-155 is clinically relevant to P. aeruginosa keratitis. We further tested miR-155 expression in a well-established murine model of P. aeruginosa keratitis. miR-155 expression was significantly enhanced in B6 mouse corneas 1, 3, and 5 days after infection (P < .001 for

all comparisons; Figure 1B), which is consistent with the clinical data. miR-155 Is Mainly Expressed in Macrophages but Not Neutrophils

To further determine the cell source of P. aeruginosa–induced miR-155 expression, we next investigated the miR-155 expression in primary PMNs and macrophages isolated from mouse bone marrow before and after P. aeruginosa infection. Polymerase chain reaction (PCR) data showed that miR-155 expression levels in bone marrow–derived PMNs were much lower than those in macrophages, with or without P. aeruginosa infection (Figure 1C). Similarly, P. aeruginosa–challenged human monocyte–derived macrophages (MDMs) displayed much higher miR-155 expression than human PMNs (Figure 1D). To further confirm the cell source of miR-155 in vivo, we sorted PMNs and macrophages from P. aeruginosa–infected mouse corneas, using flow cytometry (Figure 1E ). The expression of miR-155 in sorted PMNs (Gr-1 positive) or macrophages (F4/80 positive) was detected using single-cell PCR. Our data showed that the miR-155 in Bacterial Clearance

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Figure 1. Pseudomonas aeruginosa infection induces miR-155 (microRNA-155) expression in both human and mouse corneas and macrophages. A, miR155 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, and 5 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 murine polymorphonuclear neutrophils (PMNs) versus bone marrow–derived macrophages, human PMNs versus human monocyte–derived macrophages, murine peritoneal macrophages, and RAW264.7 cells after P. aeruginosa challenge at a multiplicity of infection of 1 for the indicated time points. E, Fluorescenceactivated 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 macrophages. Data are mean ± SEM and represent 3 individual experiments. **P < .01; ***P < .001.

expression of miR-155 in macrophages was approximately 32fold higher than that in PMNs (Figure 1F ). These results indicate that macrophages are the major inflammatory cell source of miR-155 in P. aeruginosa keratitis. We further challenged murine peritoneal macrophages (Figure 1G) and macrophage-like RAW264.7 cells (Figure 1H) with P. aeruginosa at indicated times. Real-time PCR data showed that miR-155 expression was dramatically enhanced in a time-dependent manner in both macrophages cell types. miR-155 Contributes to Corneal Susceptibility to P. aeruginosa Infection

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To investigate whether miR-155 participates in P. aeruginosa keratitis, miR-155 KO and WT B6 mice were infected with P. aeruginosa. miR-155 KO mice displayed a highly resistant phenotype against P. aeruginosa ocular infection, compared with WT B6 mice, as shown by the decreased clinical score among miR-155 KO mice 3 and 5 days after infection (P < .05 and P < .001, respectively; Figure 2A). Representative photographs of corneas from miR-155 KO and WT mice 5 days after infection are provided in Figure 2C and 2B, respectively. By 5 days after infection, miR-155 KO mice displayed less severe corneal disease (clinical score, +2/+3), whereas corneas from most WT mice were perforated (clinical score, +4). Together, these data suggest that miR-155 promotes host susceptibility to P. aeruginosa infection. miR-155 Enhances Bacterial Burden in P. aeruginosa Keratitis

Since in vivo studies indicated that deficiency of miR-155 promoted host resistance to P. aeruginosa corneal infection, we next assessed the effect of miR-155 on the bacterial component of disease pathogenesis, using a plate count assay. A reduced bacterial load was detected in the infected corneas of miR-155 KO mice, compared with WT mice, 5 days after infection (P < .01), whereas no difference between these 2 groups was detected 1 day after infection (Figure 2D). Moreover, 5 days after infection, messenger RNA levels of inducible nitric oxide synthase (iNOS) were elevated in miR-155 KO mice (P < .001), whereas levels of messenger RNA expression of the following were unchanged between the miR-155 KO and WT groups: NOX2 (NADPH oxidase), an enzyme for reactive oxygen species generation; CRAMP (cathelicidin-related antimicrobial peptide); and mBD (murine beta defensin) 2 and 3 (Figure 2E). miR-155 Inhibits Macrophage-Mediated Phagocytosis of P. aeruginosa

We further explored the mechanism of miR-155–mediated immune regulation by using murine bone marrow–derived macrophages (BMDMs) and macrophage-like RAW264.7 cells. Plate count data showed that the number of bacteria engulfed by BMDMs within 1 hour after challenge was enhanced in miR155 KO mice, compared with WT mice (Figure 3A). Moreover, phagocytosis of P. aeruginosa in miR-155–overexpressed versus 92

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Figure 2. miR-155 (microRNA-155) knockout (KO) mice are resistant to Pseudomonas aeruginosa corneal infection. miR-155 KO and wild-type B6 mice were infected with P. aeruginosa. A, Clinical score was recorded for each mouse 1, 3, and 5 days after infection. Representative photographs of infected eyes in wild-type (B) versus miR-155 KO mice (C) were taken 5 days after infection. D, Bacterial load in the infected cornea was examined by plate count in wild-type versus miR-155 KO mice 1 and 5 days after infection. E, Messenger RNA (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 KO versus wild-type mice were examined at 5 days after infection by real-time polymerase chain reaction. Data are mean ± standard error of the mean and represent 3 individual experiments, each with 5 animals per group per time per 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 demonstrated that phagocytosis of P. aeruginosa was significantly reduced after miR-155 overexpression, compared with control treatment (P < .001; Figure 3B). Immunostaining data (Figure 3C) showed less colocalization of FilmTracer Green Biofilm–labeled P. aeruginosa with Texas red–labeled cells after miR-155 mimic overexpression, compared with control

treatment, indicating that the number of P. aeruginosa phagocytosed by RAW264.7 cells was decreased by miR-155 overexpression. Results of flow cytometry (Figure 3D) also indicated that miR-155 suppressed macrophage-mediated P. aeruginosa phagocytosis by 75%, as calculated by the mean fluorescence intensity (P < .01; Figure 3E ). These data together provide evidence that miR-155 inhibits macrophage-mediated phagocytosis of P. aeruginosa. miR-155 in Bacterial Clearance

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

miR-155 Suppresses Macrophage-Mediated Bacterial Killing of P. aeruginosa

Next, we investigated the role of miR-155 in intracellular bacterial killing by means of a plate count assay. BMDMs isolated from miR-155 KO mice displayed higher capability of bacterial killing than WT mice (P < .01; Figure 4A). On the other hand, miR-155 overexpression in RAW264.7 cells significantly reduced the killing of engulfed bacteria (P < .001; Figure 4B). Moreover, NO production was significantly reduced by miR155 overexpression 12, 24, and 48 hours after P. aeruginosa challenge (P < .001 for all comparisons; Figure 4C), and iNOS expression was decreased in miR-155–overexpressed cells, 94

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compared with control RAW264.7 cells, 6, 12, 24, and 48 hours after infection (P < .01, P < .05, P < .01, and P < .01, respectively; Figure 4D). For reactive oxygen species production, no change was detected between the 2 groups (Figure 4E). These data indicate that miR-155 inhibits macrophage-mediated bacterial killing of P. aeruginosa by reducing NO production. miR-155 Suppresses Phagocytosis and Intracellular Killing by Targeting Rheb

Since the function of microRNA is largely dependent on its targets, we next searched for the specific targets of miR-155– mediated bacterial clearance. We first tested the role of

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

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

posttranscriptionally suppresses Rheb by targeting its 3′ untranslated region [18]. Western blot data showed that Rheb protein 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 phagocytosis and bacterial killing. The knockdown efficacy was confirmed by Western blot (Figure 5C). Both flow cytometry (Figure 5D) and bacteria plate count (Figure 5E) data showed miR-155 in Bacterial Clearance

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

DISCUSSION microRNAs have emerged as novel posttranslational regulators in various physiological and pathological events, such as cell proliferation, differentiation, apoptosis, and cytokine production [15, 19–21]. Nonetheless, the molecular basis involved in the microRNA-mediated ocular immune regulation remains largely unclear. Here we report an unexplored role of miR155 in regulating bacterial elimination in P. aeruginosa keratitis by targeting Rheb, which may provide a better understanding of the host antibacterial response. First, our data show that miR-155 expression is dramatically increased in both human and mouse corneas after P. aeruginosa infection. The induction of miR-155 is closely associated with infection duration, indicating the potential participation of miR155 in the progression of P. aeruginosa keratitis. Moreover, our in vivo studies show that miR-155 KO mice displayed more resistance to P. aeruginosa keratitis, compared with WT B6 mice, indicating that the presence of miR-155 contributes to the cornea’s susceptibility. Accumulating evidence demonstrates that during P. aeruginosa keratitis, bacterial virulence is the major factor leading to the disease pathogenesis. Lower bacterial load was observed in corneas from miR-155 KO mice, compared with those from WT mice, 5 days after infection, suggesting that miR-155 plays an inhibitory role in bacterial clearance. The innate immunity is the first line of host defense against invading pathogens. During P. aeruginosa keratitis, a large amount of phagocytes, including PMNs and macrophages, are rapidly recruited to the infection site and kill bacteria. It is reported that miR-155 expression is upregulated in G-CSF–mobilized CD34-positive mononuclear and neutrophil phagocyte precursors [22]. Our in vitro and in vivo studies indicate that during 96

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P. aeruginosa infection, both constitutive and inducible miR155 expression levels are much higher in macrophages than in neutrophils, suggesting that macrophages are the major cell source of P. aeruginosa–triggered miR-155 induction. Macrophages play a critical role in determining the outcome of P. aeruginosa keratitis. Studies have demonstrated that depletion of macrophages in susceptible B6 mice decreased bacterial clearance and increased the severity of P. aeruginosa keratitis [1]. Since miR-155 is mainly expressed in macrophages, its in vivo microbicidal effect is probably dependent on modulating macrophages activity. Macrophages express a series of phagocytic receptors that help to facilitate the internalization of invading pathogens. It was reported that miR-155 expression was decreased in syngeneic bone marrow transplant alveolar macrophages and that inhibition of miR-155 in alveolar macrophages increased phagocytosis of Staphylococcus aureus [23]. Our data demonstrate that miR-155 suppresses phagocytosis of P. aeruginosa in macrophages, providing further evidence regarding the role of miR-155 in bacterial clearance. Once bacteria are engulfed by macrophages, they are subjected to intracellular killing via oxygen-dependent systems, such as those involving reactive oxygen species and reactive nitrogen species, and oxygen-independent systems, such as those involving antimicrobial peptides. Hazlett et al have demonstrated that during P. aeruginosa keratitis, iNOS-derived NO is required for bacterial killing, 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 required in host resistance against P. aeruginosa corneal infection by enhancing bacterial killing and reducing inflammatory response [25, 26]. Kumar R et al reported that inhibition of miR-155 reduced the survival of Mycobacterium tuberculosis in macrophages, although the underlying mechanism of enhanced bacterial killing was not clear [27]. Our in vivo and in vitro studies demonstrate that miR-155 suppresses bacterial clearance and NO production but not reactive oxygen species or antimicrobial peptide production, indicating that miR-155 reduces intracellular killing of P. aeruginosa by inhibiting NO production. In addition, there are 2 types of macrophage activation: classical (M1) activation, which promotes bacterial killing and tissue damage, and alternative (M2) activation, which plays a critical role in tissue repair. Our study demonstrates that in the P. aeruginosa–infected macrophages, miR-155 suppresses the expression of iNOS, a well-known marker of M1 polarization, implying that miR-155 may inhibit bacterial clearance by inhibiting an M1-like macrophage activation. It is worthwhile to mention that several studies reported that human macrophages do not produce NO because of lacking exogenous tetrahydrobiopterin, an essential cofactor indispensible for iNOS activity [28–32]. Our data demonstrate that in human MDMs, P. aeruginosa infection does not induce NO production, but overexpression of miR-155 reduces the expression of NOX2, a key

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that knockdown of Rheb inhibited macrophage-mediated phagocytosis of P. aeruginosa. Moreover, iNOS expression and NO production were reduced after silencing Rheb in P. aeruginosa–challenged RAW264.7 cells (Figure 5F and 5G). Plate count data showed that bacterial killing was reduced after knockdown of Rheb (Figure 5H). To further determine whether Rheb is the major target of miR-155 in bacterial clearance, we examined the inhibitory effect of miR-155 on phagocytosis and bacterial killing in RAW264.7 cells with ectopic expression of Rheb (Figure 5I). Results of a phagocytosis assay based on bacterial plate count indicated that overexpression of miR-155 inhibited macrophage-mediated bacterial uptake in control RAW264.7 cells, but this inhibitory effect was blocked in RAW264.7 cells stably expressing Rheb (RAWRheb; Figure 5J). In addition, overexpression of miR-155 reduced iNOS expression and macrophage-mediated killing of P. aeruginosa, whereas this ability was blocked after overexpression of Rheb (Figure 5K and 5L). These data together indicate that miR-155 modulates bacterial clearance by targeting Rheb.

Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Notes Acknowledgments. We thank Dr Linda Hazlett and Dr Harley Tse from Wayne State University School of Medicine, for their useful comments and language editing. Financial support. This work was supported by the National Natural Science Foundation of China (grants U0832006 and 81261160323 [to X. H.], 31200662 and 31370868 [to M. W.], and 81172811 [to M. L.], the State Key Laboratory of Ophthalmology (Zhongshan Ophthalmic Center; Open Project Grant to X. H. and J. Y.), the Guangdong Innovative Research Team Program (2009010058 [to X. H.] and 2011Y035 [to M. W.]), the Doctoral Program of Higher Education of China (Specialized Research Funds 20100171110047 [to X. H.] and 20120171120064 [to M. W.]), the Guangdong Natural Science Foundation (10251008901000013 [to X. H.] and S2012040006680 [to M. W.]), the 111 Project (B13037 to M. W.), Guangdong Province Universities and Colleges (Pearl River Scholar Funded Scheme 2009 to X. H.), and the National Science and Technology Key Projects 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 the content of the manuscript have been disclosed.

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synthase for reactive oxygen species generation (data not shown). These data together indicated that miR-155 may use different antimicrobial mechanisms in human and murine macrophages. Studies have demonstrated that miR-155 executes different activities in various physical, pathological, and experimental conditions by repressing distinct targets, such as SOCS1 [33], C/EBP β (CCAAT/enhancer binding protein β) [34], and SHIP1 [35]. It is also reported that miR-155 decreased NO production in human umbilical vein endothelial cells and acetylcholine-induced endothelium-dependent vasorelaxation by targeting endothelial NOS [36]. However, most of the reported miR-155 targets are linked to inflammatory regulation, and nothing is known regarding the specific target involved in modulating bacterial phagocytosis and killing. Our previous study identified that miR-155 targets to the 3′ untranslated region of Rheb and suppresses Rheb expression at a posttranscriptional level [18]. In the present study, we found that regardless of the presence of P. aeruginosa infection, overexpression of miR-155 decreased, whereas inhibition of miR-155 increased the protein levels of Rheb in macrophages. Furthermore, miR-155–mediated inhibition of bacterial phagocytosis and killing are blocked in RAW264.7 cells stably expressing Rheb, indicating that Rheb is the major target of miR-155 in modulating macrophage-mediated bacterial elimination. It is reported that Rheb can directly interact with mTOR (mammalian target of rapamycin) [37] and increase mTOR activity [38]. A recent study using the same murine model of P. aeruginosa keratitis has demonstrated that inhibition of mTOR in B6 mice by treatment with rapamycin increased the clinical score and bacterial load but diminished PMN bactericidal activity in response to P. aeruginosa corneal infection [39]. Therefore, we speculate that miR-155 enhances corneal susceptibility to P. aeruginosa keratitis by targeting Rheb and suppressing bacterial clearance. We also tested the role of SHIP1, a well-established target of miR-155 [13, 27, 35] that has been reported to participate in FcR-mediated phagocytosis of immunoglobulin G (IgG)– opsonized erythrocytes [40]. Overexpression of miR-155 inhibited SHIP1 protein expression in macrophages with or without P. aeruginosa challenge. However, in our P. aeruginosa infection model, knockdown of SHIP1 expression had little effect on phagocytosis and killing of P. aeruginosa. This discrepancy may be attributed to different phagocytosis mechanisms of IgG-opsonized erythrocytes and P. aeruginosa. Collectively, our study demonstrates that miR-155 reduces macrophage-mediated bacterial clearance by targeting Rheb and thus contributes to corneal susceptibility to P. aeruginosa keratitis. To our knowledge, it is the first study to explore the role of miR-155 in P. aeruginosa killing, which may illustrate some implications for the design of microRNA-based treatment of bacterial keratitis and other infectious diseases.

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