Mammalian Target of Rapamycin Regulates IL-10 and Resistance to Pseudomonas aeruginosa Corneal Infection

Megan E. B. Foldenauer, Sharon A. McClellan, Elizabeth A. Berger and Linda D. Hazlett
J Immunol June 1, 2013, 190 (11) 5649-5658; DOI: https://doi.org/10.4049/jimmunol.1203094

Abstract

IL-10 is important in the resistance response of BALB/c mice to experimental Pseudomonas aeruginosa corneal infection. However, the cellular mechanisms by which this anti-inflammatory cytokine is regulated remain unknown. Because the mammalian target of rapamycin (mTOR) regulates IL-10 in other disease models, the present study tested its role in bacterial keratitis. After infection, corneas of rapamycin versus control-treated BALB/c mice showed worsened disease, and real-time RT-PCR confirmed that mTOR mRNA levels were significantly decreased. Rapamycin treatment also increased clinical score, polymorphonuclear neutrophil (PMN) infiltration (determined by myeloperoxidase assay), and bacterial load, but it diminished PMN bactericidal activity. Inhibition of mTOR also led to elevated mRNA and protein levels of IL-12p40, matrix metalloproteinase 9, and inducible NO synthase, whereas mRNA and protein levels of IL-10, its regulator/effector STAT-3, and suppressor of cytokine signaling 3 (a proinflammatory cytokine regulator) were decreased. Furthermore, mTOR inhibition reduced levels of proapoptotic caspase-3 and increased levels of B cell lymphoma-2 (antiapoptotic), indicative of delayed apoptosis. mTOR inhibition also altered genes related to TLR signaling, including elevation of TLR4, TLR5, and IL-1R1, with decreases in IL-1R-associated kinase 1 and an inhibitor of NF-κB, NF-κB inhibitor–like 1. Rapamycin treatment also increased levels of IFN-γ and CCAAT/enhancer binding protein, β, a gene that regulates expression of preprotachykinin-A (the precursor of substance P). Collectively, these data, as well as a rescue experiment using rIL-10 together with rapamycin, which decreased PMN in cornea, provide concrete evidence that mTOR regulates IL-10 in P. aeruginosa–induced bacterial keratitis and is critical to balancing pro- and anti-inflammatory events, resulting in better disease outcome.

Introduction

Pseudomonas aeruginosa infection in resistant Th2-responder BALB/c mice elicits production of both pro- and anti-inflammatory cytokines. Among those that are anti-inflammatory, IL-10 has been show as critical in balancing cytokines such as IFN-γ, decreasing stromal damage, and promoting successful pathogen elimination (1, 2). Nevertheless, the cellular mechanisms regulating IL-10 in the P. aeruginosa–infected cornea of BALB/c mice have not yet been determined. In this regard, in other models, the mammalian target of rapamycin (mTOR) has been of interest, as it is upstream of IL-10 (3-5) and a target of a diverse array of microbes, growth factors, hormones, and amino acids that elicit a host innate immune response.

mTOR itself is a serine-threonine kinase that also mediates cell growth and proliferation, ribosome biogenesis, and cytoskeletal organization (6, 7). It is contained within two functional complexes, mTORC1 and mTORC2, and is active when complexed with Raptor, mLST8, and PRAS40 (forming mTORC1), or when complexed with Rictor, mLST8, and Sin1 (forming mTORC2) (6, 7).

mTORC1 alone is sensitive to inhibition by the macrolide antibiotic rapamycin that blocks the formation of the complex described above. Clinically, rapamycin is used as an immunosuppressant in allogeneic transplantation. However, although used successfully in clinical practice for immunosuppression after kidney transplant (8), recent evidence suggests that it may have inflammatory side effects, including fever, anemia, and glomerulonephritis (9). Moreover, inhibition of mTOR in mice can enhance LPS-induced shock that correlates with elevated levels of proinflammatory cytokines such as IL-12p40 (10).

Similarly, another study showed that inhibition of mTOR diminished IL-10 levels but elevated IL-12p40 (and IL-23) in vitro, which was protective in vivo in experimental Listeria monocytogenes infection (5). In contrast, it has been shown that in corneal P. aeruginosa infection, IL-10 is required for resistance in the BALB/c mouse and that when macrophages (a source of the cytokine) are depleted, levels of IL-10 are decreased with concurrent elevation of IFN-γ, resulting in an overall worsening of disease (1). Polymorphonuclear neutrophils (PMN), a source of IL-12p40 (11), are of prime importance in bacterial keratitis, and when dysregulated can enhance stromal destruction and disease (11). However, the effects of rapamycin treatment on PMN remain untested and relevant to determine in bacterial keratitis.

In summary, this study provides evidence that IL-10 is regulated in the infected cornea by the mTOR pathway. This conclusion is based on data showing that inhibition of mTOR by rapamycin decreased anti-inflammatory cytokines, particularly IL-10, upregulated several proinflammatory cytokines (IL-12p40, IL-23, and IFN-γ) and their regulators, and that administration of rIL-10 with rapamycin reversed increased PMN in cornea, one effect of mTOR inhibition. Furthermore, rapamycin downregulated caspase-3 and upregulated B cell lymphoma-2 (Bcl-2), with the latter prolonging inflammatory cell viability, possibly further contributing to stromal destruction and unresolved disease.

Materials and Methods

Mice

Female 8-wk-old BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in accordance with the National Institutes of Health guidelines. The animals were treated humanely in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

Bacterial culture and infection

P. aeruginosa cultures (strain 19660; purchased from the American Type Culture Collection, Manassas, VA) were grown in peptone tryptic soy broth (PTSB) at 37°C on a 150 rpm rotary shaker for 18 h to an OD reading between 1.3 and 1.8 at 540 nm. Stock cultures were centrifuged at 6000 × g for 10 min, washed once with sterile saline, recentrifuged, and resuspended to a final concentration of ∼1 × 106 CFU/μl (1). Under a stereoscopic microscope (magnification, ×40), a sterile 25⅝-gauge needle was used to scar the left central cornea of ethyl ether–anesthetized BALB/c mice, after which a 5 μl suspension of P. aeruginosa was topically applied. To analyze phagocytic capacity, GFP-expressing P. aeruginosa was grown to a logarithmic phase in PTSB with kanamycin (50 μg/ml), washed twice with PBS, resuspended and diluted in PTSB (with kanamycin), and used as described below for phagocytosis assays.

Ocular response to bacterial infection

Corneal disease was graded using an established scale (12): 0, clear or slight opacity, partially or fully covering the pupil; 1, slight opacity, fully covering the anterior segment; 2, dense opacity, partially or fully covering the pupil; 3, dense opacity, covering the entire anterior segment; and 4, corneal perforation. After infection, a clinical score was recorded (days 1, 3, and 5) for each group of mice (n = 5/group/treatment/assay). Lastly, representative corneas were photographed using a slit lamp to document the effects of rapamycin versus control treatment.

Rapamycin treatment

Rapamycin (LC Laboratories, Woburn, MA) was prepared to a concentration of 20 μg/μl in 100% ethanol and stored at −20°C. Before i.p. injection, the rapamycin in ethanol was diluted in sterile PBS. To test the appropriate concentration of rapamycin, BALB/c mice (n = 5/group/time/assay) were anesthetized with ether and i.p. injected with 100 μl rapamycin (1.5 or 3.0 mg/kg) (5) or sterile PBS (Mediatech, Manassas, VA) on the day before infection (day −1) and each day through 5 d postinfection (p.i.). Results indicated that the higher concentration of rapamycin significantly lowered IL-10 protein levels (comparative data not shown), and thus all subsequent experiments were done using the higher concentration of rapamycin prepared as described above. In a separate experiment BALB/c mice were injected with rapamycin as described above with or without injection of rIL-10 (R&D Systems, Minneapolis, MN). rIL-10 (1 μg/μl) was injected subconjunctivally the day before infection and a similar amount was injected i.p. on the day of infection and at 1 d p.i. Corneas were harvested for myeloperoxidase (MPO) assay at 1 and 3 d p.i. as described below.

Quantification of corneal PMN

An MPO assay was used to quantitate the PMN leukocyte infiltrate in the corneas of rapamycin and PBS-treated BALB/c mice (n = 5/group/time) at 1 and 3 d p.i. Corneas were harvested, homogenized in 1 ml potassium phosphate buffer (50 mM, pH 6.0) containing 0.5% hexodecyltrimethylammonium bromide (Sigma-Aldrich) using glass microtissue grinders. The samples were freeze-thawed four times and centrifuged at 14,000 rpm for 10 min. To measure MPO, a 100 μl sample of supernatant was added to fresh 2.9 ml o-dianisidine dihydrochloride substrate buffer (16.7 mg/ml; Sigma-Aldrich) with 0.0005% hydrogen peroxide. The change in absorbance at 460 nm was read every 30 s for 5 min on a Heλios α spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Afterward, units of MPO per cornea were calculated: one unit of MPO activity is equivalent to ∼2 × 105 PMN/μl (13).

Quantification of viable bacteria

Bacteria were quantitated at 1 and 3 d p.i. in individual infected corneas of BALB/c mice (n = 5/group/time) after rapamycin or PBS treatment. Each cornea was homogenized in 1 ml sterile PBS containing 0.25% BSA; 100 μl corneal homogenate was serially diluted 1:10 in the same solution. Selected dilutions were plated in triplicate on Pseudomonas isolation agar plates (Becton Dickinson, Franklin Lakes, NJ). Plates were incubated overnight at 37°C and the number of viable bacteria manually counted. Results are reported as 1 × 103 CFU/cornea ± SEM.

Quantification of PMN phagocytic and intracellular killing capacity

PMN from BALB/c mice were isolated (14) and processed essentially as described before (15). Briefly, mice received an i.p. injection of a 9% casein solution (1.0 ml) and a second similar injection 24 h later. PMN (90% of elicited cells) were lavaged from the peritoneal cavity 3 h after the second injection, washed, and isolated using a Percoll gradient. Viable cells (≥94.3%) were stained with trypan blue and counted using Cellometer Vision CBA analysis system version 2.1.0. (Nexcelom Bioscience, Lawrence, MA) and resuspended in RPMI 1640 (with 10% FBS) at the desired concentration for in vitro stimulation. Cells were treated with rapamycin (0.01 and 1.0 μM) or sterile 1× PBS (negative and positive controls) for 18 h at 37°C. Cells were washed to remove the inhibitor, then resuspended in media. PMN viability after rapamycin treatment was decreased from ≥94.3% to between 87.5 and 90.4%.

For phagocytosis assays (16), 1 × 106 CFU/μl GFP-labeled bacteria was added to each well (a ratio of 2.5:1, bacteria to PMN) of a 12-well plate, omitting bacterial addition to the negative control wells. Plates were centrifuged at 500 × g for 10 min and then incubated for 2 h at 37°C. Cells were washed twice to remove bacteria, then incubated with 0.04% trypan blue to quench the fluorescence of surface-associated bacteria and to again measure PMN viability (≥87.5%).

To analyze intracellular killing, PMN were incubated with P. aeruginosa, harvested, and treated as above. Then, after 1 h incubation at 37°C, 200 μg/ml gentamicin (17) was added for an additional 3 h to ensure killing of extracellular bacteria. Cells were washed twice with HBSS. Supernatant was removed and 0.5 ml ice-cold water was added to lyse PMN. Samples were kept on ice for 10 min, after which 0.5 ml 2× HBSS was added to a final volume of 1 ml. Cell suspension (250 μl) was plated in triplicate on Pseudomonas isolation agar (Becton Dickinson) and incubated for 24 h at 37°C. Afterward, colonies were manually counted and recorded as the mean CFU ± SEM. The experiment was repeated twice.

Real-time RT-PCR

Mice were sacrificed and normal and infected corneas from rapamycin- and PBS-treated mice (n = 5/group/time) were removed (1, 3, and 5 d p.i.). Individual corneas were briefly stored in RNA STAT-60 (Tel-Test, Friendsville, TX) at −20°C before processing. Total corneal RNA was extracted with chloroform (200 μl/ml RNA STAT-60; Sigma-Aldrich) and precipitated overnight with isopropanol (500 μl/ml RNA STAT-60; Sigma-Aldrich). Samples were washed in cold 75% ethanol. To produce a cDNA template for the PCR reaction, 1 μg each RNA sample was reverse transcribed using Moloney murine leukemia virus. The 20 μl mixture contained 200 U Moloney murine leukemia virus reverse transcriptase, 10 U RNase inhibitor, 500 ng oligo(dT) primers, 10 mM dNTPs, 100 mM DTT, and Moloney murine leukemia virus reaction buffer (all from Invitrogen, Carlsbad, CA). cDNA products were diluted 1:25 with diethylpyrocarbonate-treated water, and a 2 μl cDNA aliquot was used for real-time RT-PCR. mRNA levels of mTOR, STAT-3, IL-10, IL-12p40, IL-23, inducible NO synthase (iNOS), matrix metallopeptidase (MMP)-9, suppressor of cytokine signaling (SOCS)3, caspase-3, Bcl-2, CCAAT/enhancer binding protein, β (Cebpb), NF-κB inhibitor–like 1 (NFκBIL-1), TLR4, TLR5, IFN-γ, IL-1R1, and IL-1R–associated kinase (IRAK)-1 were tested by real-time RT-PCR (MyiQ single color real-time PCR detection system; Bio-Rad, Hercules, CA). Real-Time SYBR Green/Fluorescein PCR Master Mix (Bio-Rad) was used for the PCR reaction with primer concentrations of 10 μM. After a preprogrammed hot start cycle (3 min at 95°C), the parameters used for PCR amplification were 15 s at 95°C and 60 s at 60°C with the cycles repeated 45 times. The fold differences in gene expression were calculated after normalization to β-actin and are expressed as the relative mRNA concentration ± SEM. The primer pair sequences used for real-time RT-PCR are shown in Table I.

ELISA

After mice were treated with rapamycin or sterile PBS, IL-10, IL-12p40, p-STAT-3, Cebpb, and IFN-γ protein levels were determined in normal and infected mouse corneas (n = 5/group/time, repeated once) using ELISA kits (R&D Systems; except Cebpb, which was purchased through Abs-online.com, Atlanta, GA). For these assays, tissue was prepared as described before (2). Fifty- (IL-12p40, IFN-γ) or 100-μl (IL-10, p-STAT-3, and Cebpb) aliquots of each supernatant were assayed in duplicate according to the manufacturer’s instructions (R&D Systems; Cebpb was from Abs-online.com). Sensitivities for the assays were >4.0 (IL-10), 0.6–2.7 (IL-12p40), 33 (Cebpb), >2.0 (IFN-γ), and >4.48 pg/ml (p-STAT-3) (R&D Systems). Results are expressed as picograms per milliliter (femtograms per milliliter only for IFN-γ) of protein ± SEM.

Immunohistochemistry

After sacrificing mice, normal and 1 and 5 d p.i. eyes were enucleated from rapamycin- or PBS-treated animals (n = 5/group/time), embedded in OCT compound (Sakura Finetek, Torrance, CA) and snap frozen in liquid nitrogen. Sections (10 μm) were cut, fixed in acetone, and incubated in blocking agent as described before (1). Sections were simultaneously incubated with individual primary Abs: rabbit anti-mouse caspase-3 (1:800; Cell Signaling Technology, Danvers, MA) and hamster anti-mouse Bcl-2 (1:100; BD Biosciences, San Jose, CA) in blocking agent for 1 h. Sections were then rinsed with phosphate buffer and incubated for another hour with Alexa Fluor 633 goat anti-rabbit secondary Ab for caspase-3 (1:1500; Invitrogen) and Alexa Fluor 546 goat anti-hamster for Bcl-2 (1:1500; Molecular Probes, Eugene, OR) in 0.0 1M Tris-HCl buffer. Sections were rinsed in the latter buffer and then incubated with nuclear acid stain (Sytox Green, 1:20,000; Lonza, Walkersville, MD) and rinsed again. Digital images were captured on a confocal laser-scanning microscope (TCS SP2; Leica Microsystems, Exton, PA). Negative controls for each group were processed similarly, with the exception that each primary Ab was replaced by the same host species IgG.

Nitrite concentration (Griess assay)

NO production was measured in uninfected corneas and 1 and 5 d p.i. after PBS versus rapamycin treatment (n = 5/group/time) using Griess reagent (Sigma-Aldrich), which quantifies the stable end product nitrite. Corneal samples were homogenized in sterile PBS with microtissue grinders and 100-μl aliquots were incubated in 100 μl Griess reagent for 15 min at room temperature. Absorbance was measured at 570 nm, and nitrite concentrations were calculated using a sodium nitrite standard curve. The data are reported as the mean cornea concentration of nitrite (μM) ± SEM.

TLR PCR array

The corneas of rapamycin- versus PBS-treated mice (n = 5/group/time) were harvested at 3 d p.i., pooled, and processed for cDNA using an RT2 First Strand Kit (Qiagen, Valencia, CA). mRNA levels of 84 genes were profiled using a TLR RT2 Profiler PCR Array (Qiagen). Selected genes that had at least a 2-fold or greater change were tested by real-time RT-PCR and ELISA assay.

Statistical analysis

The difference in clinical score between two groups was tested by the Mann–Whitney U test. For all other experiments, an unpaired, two-tailed Student t test (for comparisons between two groups) was used. All data were considered significant at p ≤ 0.05. Each experiment was repeated at least once to ensure reproducibility, and data from a representative experiment are shown. Quantitative data are expressed as the mean ± the SEM, except for the clinical score data in which medians are shown.

Results

Disease response, mTOR inhibition, and PMN infiltration

More severe disease (Fig. 1A) was seen after rapamycin treatment at 3 and 5 d p.i. (p = 0.02 for both) when compared with PBS-treated controls; no significant difference was observed at 1 d p.i. Photographs taken with a slit lamp of representative eyes from both groups at 5 d p.i. (Fig. 1B, 1C) confirmed that rapamycin treatment increased corneal opacity and resulted in corneal thinning (Fig. 1B), whereas the cornea of PBS-treated mice exhibited slight central opacity (Fig. 1C). mTOR mRNA levels also were tested using real-time RT-PCR (Fig. 1D). Rapamycin downregulated mTOR when compared with PBS treatment in the normal, uninfected cornea and at 1 and 5 d p.i. (p = 0.0004, p = 0.05, p = 0.04). An MPO assay was used to quantitate PMN in the infected cornea of both groups (Fig. 1E) and revealed that after rapamycin versus PBS treatment, MPO levels were significantly increased at 3 d p.i. (p = 0.007) but did not differ between groups at 1 d p.i.

FIGURE 1.
FIGURE 1.

Effects of rapamycin treatment in vivo. Clinical scores (A) were significantly increased at 3 and 5 d p.i. in rapamycin- versus PBS-treated mice. Photographs with a slit lamp confirmed that the cornea of rapamycin- (B) versus PBS- (C) treated mice had increased opacity and corneal thinning. Rapamycin versus PBS also significantly decreased mTOR mRNA levels (D) in normal uninfected cornea, and at 1 and 5 d p.i. MPO levels (E) were significantly elevated at 3 d p.i. in rapamycin- versus PBS-treated mice, with no differences at 1 d p.i. Bars show median values. Original magnification ×7.

Bacterial load, PMN phagocytosis and intracellular killing

Viable bacterial load in the cornea of rapamycin- versus PBS-treated mice was significantly higher at 3 d p.i. (Fig. 2A; p < 0.0001); no significant difference was observed at 1 d p.i. An in vitro PMN phagocytosis assay (Fig. 2B) compared PBS with no bacteria (negative control), with GFP-labeled bacteria (positive control), and with GFP-labeled bacteria with rapamycin at either 0.01 or 1.0 μM concentration. Rapamycin at 1.0 μM concentration significantly increased the percentage of PMN containing GFP-labeled bacteria (p = 0.002) over the positive control. However, no differences were detected between the positive control and the lower concentration of rapamycin. The ability of PMN to kill bacteria was similarly assessed among the groups and was significantly compromised (Fig. 2C; p = 0.04) compared with the positive control only at a concentration of 1.0 μM rapamycin.

FIGURE 2.
FIGURE 2.

Rapamycin effects on PMN phagocytosis and intracellular killing. (A) Bacterial load was significantly elevated at 3 d p.i. in rapamycin- versus PBS-treated mice, with no differences at 1 d p.i. (B) Rapamycin treatment (1.0 μM) significantly increased PMN phagocytosis when comparing the percentage of cells containing GFP+ bacteria with the positive control. No effect was seen at the lower concentration (0.01 μM). (C) Plate counts assessed bacterial killing and only 1.0 μM rapamycin showed a significant increase in bacterial load (decreased killing) compared with the positive control group.

Real-time RT-PCR of rapamycin effects on STAT-3, IL-10, IL-12p40, and IL-23 mRNA levels

The balance between pro- and anti-inflammatory cytokines is critical to disease resistance in the infected cornea of BALB/c mice (1, 2). Thus, real-time RT-PCR was used to characterize the role of mTOR in their regulation. Fig. 3A, 3C, 3E, and 3G show corneal mRNA levels of STAT-3, IL-10, IL-12p40, and IL-23 at various times after rapamycin or PBS treatment. Rapamycin treatment resulted in a significant increase in STAT-3 mRNA expression (Fig. 3A) in the normal cornea and a decrease at 3 and 5 d p.i. (p < 0.0001, p < 0.0001, p = 0.05) when compared with PBS controls. No difference was detected at 1 d p.i. between the two groups. IL-10 mRNA expression (Fig. 3C) was significantly decreased at 1 and 5 d p.i. (p = 0.03 and p = 0.04) following rapamycin treatment, with no difference between groups in the normal cornea or at 3 d p.i. In contrast, IL-12p40 (Fig. 3E) mRNA was significantly increased after rapamycin treatment at 1 and 3 d p.i. (p = 0.009 and p < 0.0001), with no difference between groups in the normal cornea or at 5 d p.i. mRNA levels of IL-23 (Fig. 3G) also were increased significantly in normal cornea and at 1 and 5 d p.i. (p = 0.0003, p < 0.0001 for both times p.i.).

FIGURE 3.
FIGURE 3.

Effects of rapamycin treatment on STAT-3, IL-10, and IL-12 expression levels in cornea and rIL-10 rescue experiment. After rapamycin treatment: STAT-3 mRNA (A) levels were downregulated at 3 and 5 d p.i., but increased in the normal uninfected cornea and unchanged at 1 d p.i.; mRNA levels of IL-10 (C) were decreased at 1 and 5 d p.i., undetected in the normal uninfected eye, and not different between groups at 3 d p.i.; IL-12p40 (E) mRNA levels were increased at 1 and 3 d p.i. but did not differ between groups at 5 d p.i. or in the normal cornea. IL-23 mRNA (G) was increased after rapamycin treatment in normal cornea and at 1 and 5 d p.i. For protein, p-STAT-3 (B) was downregulated after rapamycin treatment in normal cornea and at 1 and 3 d p.i. IL-10 protein levels (D) were not detected in either group in normal cornea and did not differ at 1 d p.i. between groups, but they decreased after rapamycin treatment at 5 d p.i. IL-12 p40 protein (F) was increased at 3 d p.i. but did not differ between groups in the normal cornea or at 1 d p.i. Injection of rIL-10 (H) together with rapamycin versus PBS treatment decreased PMN (MPO assay) at 3 but not 1 d p.i.

Rapamycin treatment leads to a proinflammatory environment, but rIL-10 reverses rapamycin elevation of MPO

To selectively confirm the real-time RT-PCR data, ELISA assays were performed to determine the protein levels of p-STAT-3, IL-10, and IL-12p40 (Fig. 3B, 3D, 3F). Rapamycin treatment significantly decreased levels of p-STAT-3 (Fig. 3B), the activated form of STAT-3, in the normal cornea and at 1 and 5 d p.i. (p = 0.006, p = 0.03, p = 0.02). In the normal cornea, IL-10 protein levels were below detectability for both groups (Fig. 3D) and did not differ between groups at 1 d p.i. However, at 5 d p.i., rapamycin decreased IL-10 levels compared with control treatment (p = 0.03). In contrast, IL-12p40 (Fig. 3F) was increased in the rapamycin-treated group at 3 d p.i. (p = 0.03), with no differences seen between groups in the normal cornea or 1 d p.i. Lastly, when rIL-10 was injected with rapamycin versus rapamycin treatment alone (Fig. 3H), MPO activity was decreased, reversing the effects of rapamycin at 3 d p.i. (p = 0.01), but not at 1 d p.i.

Real-time RT-PCR testing downstream effectors of IL-10

To explore the impact of IL-10 downregulation, real-time RT-PCR (Table I) was used to test several downstream effectors of IL-10: SOCS3, MMP-9, and iNOS (Fig. 4A–C). Treatment with rapamycin significantly decreased mRNA levels of the proinflammatory cytokine regulator SOCS3 (Fig. 4A) at both 1 and 5 d p.i. (p < 0.0001 and p = 0.03) compared with PBS controls, with no difference between groups in the normal cornea. In contrast, rapamycin treatment significantly increased levels of MMP-9 at 5 d p.i. (Fig. 4B; p = 0.05), with no differences between groups in the normal cornea or at 1 d p.i. Furthermore, the NO catalyzing enzyme iNOS was significantly upregulated after rapamycin versus PBS treatment (Fig. 4C) in the normal cornea and at both 1 and 5 d p.i. (p = 0.01, p = 0.02, p = 0.02).

View this table:
Table I. Nucleotide sequences of mouse primers for real-time RT-PCR
FIGURE 4.
FIGURE 4.

Response of IL-10 effectors to rapamycin treatment. mRNA levels for SOCS3 (A) were significantly decreased at 1 and 5 d p.i. after rapamycin versus PBS treatment. mRNA levels of MMP-9 (B) were upregulated at 5 d p.i., whereas iNOS mRNA levels (C) were significantly increased in normal cornea and at 1 and 5 d p.i. Nitrite levels (D) were significantly increased in the rapamycin-treated group only at 5 d p.i.

Griess analysis of nitrite production

Levels of NO were tested using a Griess assay that measures nitrite concentration (Fig. 4D). At 1 d p.i., there was no significant difference in corneal nitrite levels between PBS- and rapamycin-treated mice. However, by 5 d p.i., nitrite levels were significantly increased in the rapamycin-treated group (p = 0.02).

RT2 Profiler PCR array and real-time RT-PCR of TLR-related genes

TLR-related genes were profiled in the cornea at 3 d p.i. using a PCR array. Several genes with >2-fold change in expression were selected for further analysis using real-time RT-PCR (labeled in Fig. 5A and shown in Table II): TLR4, TLR5, IRAK-1, NFκBIL-1, IL-1R1, IFN-γ, and Cebpb. Rapamycin treatment significantly increased mRNA expression levels of TLR4 in the normal cornea and at 1 and 5 d p.i. (Fig. 5B; p = 0.002, p = 0.02, p = 0.02). TLR5 mRNA levels also were increased in the normal cornea and at 1 d p.i. (Fig. 5C; p = 0.004, p = 0.003) with no significant change at 5 d p.i. IRAK-1, whose signaling leads to the translocation of NF-κB from the cytoplasm to the nucleus, was significantly upregulated with rapamycin treatment in the normal cornea (Fig. 5D; p = 0.009), but by 5 d p.i., levels were downregulated (Fig. 5D; p = 0.002). IL-1R1 was significantly increased by rapamycin treatment in the normal cornea and at 1 d p.i. (Fig. 5E; p < 0.0001 and p = 0.009). No significant difference was observed at 5 d p.i. Finally, an inhibitor of NF-κB translocation to the nucleus, NFκBIL-1, was significantly downregulated with rapamycin treatment at 1 and 5 d p.i. but was upregulated in the normal cornea (Fig. 5F; p = 0.04, p < 0.0001, p = 0.03).

FIGURE 5.
FIGURE 5.

TLR RT2 Profiler PCR array and real-time RT-PCR comparing rapamycin to PBS treatment. Scatter plots of several genes from PCR array that exhibited at least a 2-fold change at 3 d p.i. are shown (A). These also were tested by RT-PCR: mRNA levels for TLR4 (B) were elevated in the normal uninfected cornea and at 1 and 5 d p.i; TLR5 mRNA levels (C) were upregulated in normal cornea and at 1 d, but not 5 d p.i. IRAK-1 levels (D) were elevated in normal cornea, unchanged at 1 d p.i., and decreased at 5 d p.i. For IL-1R1 (E), levels were increased in the normal cornea and at 1 d p.i. but were not significantly different at 5 d p.i. For NFκBIL-1 (F), levels were increased in normal cornea, but decreased at 1 and 5 d p.i.

View this table:
Table II. Selected TLRs from the RT2 Profiler PCR array

Rapamycin increases proinflammatory genes Cebpb and IFN-γ

Rapamycin treatment significantly increased mRNA levels of proinflammatory Cebpb in the normal cornea and at 1 and 5 d p.i. (Fig. 6A; p < 0.0001, p = 0.001, p = 0.002). Likewise, IFN-γ was upregulated in the rapamycin-treated group in the normal cornea and at 1 and 5 d p.i. (Fig. 6C; p = 0.02, p = 0.04, p = 0.004). Rapamycin inhibition of mTOR also resulted in an increase in protein levels of Cebpb at 1 and 5 d p.i. (Fig. 6B; p = 0.02, p < 0.0001). It also resulted in increased IFN-γ expression at 6 d p.i., with no difference between groups at 1 d p.i. (Fig. 6D)

FIGURE 6.
FIGURE 6.

Effect of rapamycin versus PBS treatment on proinflammatory cytokines. Rapamycin treatment significantly increased Cebpb mRNA (A) and IFN-γ (C) in the normal cornea and at 1 and 5 d p.i. Cebpb protein levels (B) were increased at 1 and 5 d p.i. but did not differ between groups in the normal cornea. IFN-γ protein (D) was significantly increased at 6 d p.i. but was undetectable at 1 d p.i. in either group.

Rapamycin increases pro- versus antiapoptotic genes

To test the effects of rapamycin treatment on apoptosis, caspase-3 and Bcl-2 were assayed by real-time RT-PCR and immunostaining. Caspase-3 (Fig. 7A) was significantly decreased in the normal cornea of rapamycin- versus PBS-treated group and at 1 d p.i. (p = 0.04 for both). However, no significant differences between groups were seen at day 3 p.i. Bcl-2 was significantly increased in the normal cornea and at 1 and 3 d p.i. (Fig. 7B; p = 0.0001, p = 0.003, and p < 0.0001).

FIGURE 7.
FIGURE 7.

Effect of rapamycin treatment on apoptosis. Rapamycin versus PBS treatment: (A) significantly downregulated caspase-3 mRNA in the normal cornea and at 1 d p.i. with no differences between groups at 3 d p.i.; (B) Bcl-2 mRNA was upregulated in normal cornea and at 1 and 3 d p.i. (C) Confocal images show a decrease in caspase-3 (blue) and increase in Bcl-2 (red) at 1 and 5 d p.i. in the cornea of rapamycin versus PBS treated mice. Controls in which the primary Ab was replaced with same host species IgG were negative. Sytox Green (green) was used for nuclear labeling. Original magnification ×208.

Qualitative differences in immunostaining for caspase-3 (blue) and Bcl-2 (red) were detected between rapamycin- versus PBS-treated groups (Fig. 7C) at 1 and 5 d p.i. Bcl-2 staining was markedly more intense at 1 and 5 d p.i. in the rapamycin-treated group whereas caspase-3 was barely detectable when compared with PBS controls. In the PBS group, staining for caspase-3 was increased at 1 and 5 d p.i., whereas Bcl-2 staining was markedly reduced. Representative same host species IgG negative controls are shown for the rapamycin- and PBS-treated groups.

Discussion

Previous study has shown that in P. aeruginosa–induced keratitis, macrophages restrict bacterial growth, limit PMN influx, and upregulate IL-10, which is critical to balancing/downregulating proinflammatory cytokines such as IFN-γ, and that these contribute to the resistance response of BALB/c mice (1). The present work expands upon these findings by examining the relationship between IL-10 and its regulation by mTOR in the infected BALB/c cornea.

mTOR is an upstream regulator of immunoregulatory cytokines (e.g., IL-10) in other disease paradigms and is implicated in both growth and inflammatory pathways (3, 5). Previous work also has shown that mTOR plays a role in regulating the IL-10/IL-12 axis in LPS-stimulated macrophages and controls the innate immune response (4).

The present work has specifically demonstrated the adverse effects of rapamycin treatment after P. aeruginosa infection in the cornea. The data provide evidence that decreased IL-10 and increased IL-12p40 and IL-23 levels are the foundation for these effects. Although proinflammatory cytokines such as IL-12p40 are essential to amplify the innate immune response, without anti-inflammatory cytokines such as IL-10 counterbalancing these effects, the inflammatory response is unchecked (4), as seen in the present study. In fact, a rescue experiment, that is, the injection of rIL-10 together with rapamycin versus rapamycin treatment alone, revealed decreased PMN number in the cornea. These data provide concrete evidence of at least one mechanism by which IL-10 participates to balance inflammatory responses in the cornea.

Inhibition of mTOR gene activation is a common technique used to characterize the function of mTOR. Rapamycin is an often used inhibitor that acts at this level (3–7) and has been shown to downregulate mRNA levels of mTOR in other systems (18), supportive of our findings in the keratitis model. Rapamycin is used clinically for immunosuppression after kidney transplant, and inflammatory conditions are not routinely seen (8). Additionally, at the concentration used in the present study, others (19) have shown that rapamycin levels in blood had therapeutic immunosuppressive effects in animal models of graft-versus-host disease. Nonetheless, Säemann et al. (9) reviewed the role of mTOR inhibition by rapamycin in transplant immunity and found that, despite preliminary benefits to prevent rejection, side effects accompanied its use, including rash, fever, pneumonitis, glomerulonephritis, and anemia. Another study showed that administration of rapamycin increased the lethality of endotoxin (LPS)-mediated shock in mice (10) and resistance to antimicrobial therapy (20).

These effects suggest that inhibition of mTOR has profound impact on elements of the inflammatory response, including infiltrating cells such as PMN. In this regard, we have shown that rapamycin increased PMN number in the infected cornea. This implies that bacterial clearance should be enhanced; however, viable bacterial plate counts were increased when compared with controls. Thus, the phagocytic and bactericidal capacity of these cells to engulf and kill P. aeruginosa was examined. The ability of mouse PMN to phagocytize bacteria was enhanced at the higher concentration (1.0 μM) of rapamycin used; however, the higher concentration of rapamycin decreased intracellular killing capacity. These data are somewhat consistent with other work that examined the effects of rapamycin (concentration of 10–20 nM) on human PMN function (in cirrhosis patients), and determined that inhibition of mTOR decreased PMN bactericidal activity (21), but not uptake. Differences between their data and ours could reflect our usage of murine versus human cells and that we used a higher concentration of rapamycin.

Furthermore, our data show that rapamycin treatment led to upregulated deleterious downstream effectors of IL-10 (e.g., MMP-9, iNOS) and to downregulated cytokines that participate in control of the inflammatory response (e.g., SOCS3, STAT-3). The data also are in agreement with past studies in that others have shown that SOCS3 is a mediator of LPS-stimulated IL-10 release in response to infection (22). Additionally, SOCS3-deficient mice are vulnerable to enhanced expression of Th1 inflammatory cytokines, promoting infiltration of inflammatory cells (e.g., PMN, macrophages, T cells) (23).

Previously, our laboratory demonstrated that rMMP-9–treated BALB/c mice exhibited worsened disease based on increased clinical disease score, MPO, Langerhans cell number, and protein levels of proinflammatory cytokines (24). MMP-9 is among the most widely studied MMP in the cornea, as it preferentially degrades basement membrane components such as type IV collagen. In this study, loss of mTOR signaling led to an increase in MMP-9 mRNA. Furthermore, PMN are a source of matrix metalloproteinases (24), and because rapamycin increased the number of these cells in the cornea after infection, it is likely that they contribute to elevated levels of MMP-9. Ultimately, this increase fosters destruction of the corneal stromal cytoarchitecture and decreased ability for tissue restoration (24).

Berlato et al. (22) observed a correlation between decreased iNOS levels and cells that were treated with IL-10. This is consistent with our data, as we have shown that inhibition of mTOR elevates iNOS and nitrite levels. In previous reports, our laboratory has shown that iNOS is constitutively expressed in the BALB/c cornea and is required for bacterial killing (25). However, overproduction of iNOS-derived NO (through upregulation of IFN-γ) is associated with susceptibility to P. aeruginosa infection (26) and these past data are consistent with current studies.

In this study, we establish that rapamycin affects numerous elements of the TLR pathway that are instrumental in the response to P. aeruginosa infection. Others have characterized TLR4 and TLR5 as regulators of P. aeruginosa–induced keratitis in macrophages (27). In our laboratory, we have demonstrated that TLR4 is constitutively produced and activated in the BALB/c cornea after infection (28) and is required for resistance against P. aeruginosa. Its signaling cascade includes IRAK-1 and leads to the translocation of NF-κB from the cellular cytoplasm to the nucleus, causing the transcription of proinflammatory genes such as IFN-γ and IL-1β (whose receptor, IL-1R1, is also upregulated by rapamycin). In the present study, we have shown that rapamycin-treated mice have increased levels of TLR4, TLR5, and IRAK-1 mRNA, indicating increased stimulation of the proinflammatory pathway. Moreover, we also demonstrated that an inhibitor of NF-κB translocation, NFκBIL-1, is downregulated in response to rapamycin treatment.

Cebpb increases expression of the ppt-A gene that gives rise to substance P (SP) (29), a proinflammatory neuropeptide that worsens bacterial keratitis in BALB/c mice (2). In fact, preliminary data suggest that rapamycin and SP elicit a similar pattern of disease response in the cornea of P. aeruginosa–infected BALB/c mice, with a similar outcome (30). In this study, we have demonstrated that Cebpb is significantly upregulated in rapamycin-treated mice (mRNA, protein); however, SP regulation by Cebpb in the cornea has been untested and may require further examination.

Lastly, tight regulation of the balance between apoptosis and cell survival, as well as the timing of apoptotic events, is critical to immune defense (12, 31). Others have shown that persistence of apoptotic cells can worsen disease outcome (12, 31). In the susceptible C57BL/6 mouse, we have shown that PMN have delayed apoptosis that contributes to inflammation-induced tissue damage (12). In the same study, administration of SP to resistant BALB/c mice delayed apoptosis, leading to a similar outcome of worsened disease. Another study reported that in a mouse model of LPS-induced acute lung injury, delay of apoptosis also prolonged the inflammatory response (32). Similarly, treatment with rapamycin as we have reported delayed apoptosis, which again is consistent with past studies and with the poor outcome of enhanced disease after infection.

In summary, inhibition of mTOR by rapamycin treatment increased disease in the cornea of resistant BALB/c mice through downregulation of the anti-inflammatory cytokine IL-10 and upregulation of proinflammatory IL-12p40 and IL-23. Furthermore, the absence of IL-10 led to dysregulation of several downstream effectors (MMP-9, SOCS3, STAT-3, and iNOS) and elements of the TLR pathway (TLR4, TLR5, IRAK-1, IL-1R1, NFκBIL-1, Cebpb, and IFN-γ). Moreover, we have demonstrated that loss of mTOR signaling generates an antiapoptotic corneal environment in which damaged and dying cells could contribute to further tissue destruction. Rapamycin treatment also increased PMN number but reduced their capacity for intracellular killing. Lastly, rIL-10 together with rapamycin injection was capable of reducing PMN number, strengthening the tenet held in this study that IL-10 is critical to disease resolution. Collectively, these data provide evidence that loss of mTOR signaling elicits a dysregulated proinflammatory response in the infected BALB/c mouse cornea that exacerbates P. aeruginosa–induced keratitis.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Eye Institute/National Institutes of Health Grants R01 EY002986, R01 EY016058, and P30 EY004068 (to L.D.H.).

  • Abbreviations used in this article:

    Bcl-2
    B cell lymphoma-2
    Cebpb
    CCAAT/enhancer binding protein, β
    iNOS
    inducible nitric oxide synthase
    IRAK
    IL-1R–associated kinase
    MMP
    matrix metallopeptidase
    MPO
    myeloperoxidase
    mTOR
    mammalian target of rapamycin
    mTORC
    mammalian target of rapamycin complex
    NFκBIL-1
    NF-κB inhibitor–like 1
    p.i.
    postinfection
    PMN
    polymorphonuclear neutrophil
    PTSB
    peptone tryptic soy broth
    SOCS
    suppressor of cytokine signaling
    SP
    substance P.

  • Received November 7, 2012.
  • Accepted March 22, 2013.

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