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. Author manuscript; available in PMC: 2013 Feb 28.
Published in final edited form as: J Leukoc Biol. 2008 Apr 17;83(6):1512–1521. doi: 10.1189/jlb.0108076

Inhibition of corneal inflammation by liposomal delivery of short-chain, C-6 ceramide

Yan Sun *, Todd Fox †, Gautam Adhikary *, Mark Kester †, Eric Pearlman *,1
PMCID: PMC3584160  NIHMSID: NIHMS387117  PMID: 18372342

Abstract

Ceramide is recognized as an antiproliferative and proapoptotic sphingolipid metabolite; however, the role of ceramide in inflammation is not well understood. To determine the role of C6-ceramide in regulating inflammatory responses, human corneal epithelial cells were treated with C6-ceramide in 80 nm diameter nano-liposome bilayer formulation (Lip-C6) prior to stimulation with UV-killed Staphylococcus aureus. Lip-C6 (5 μM) inhibited the phosphorylation of proinflammatory and proapoptotic MAP kinases JNK and p38 and production of neutrophil chemotactic cytokines CXCL1, CXCL5, and CXCL8. Lip-C6 also blocked CXC chemokine production by human and murine neutrophils. To determine the effect of Lip-C6 in vivo, a murine model of corneal inflammation was used in which LPS or S. aureus added to the abraded corneal surface induces neutrophil infiltration to the corneal stroma, resulting in increased corneal haze. Mice were treated topically with 2 nMoles (811 ng) Lip-C6 or with control liposomes prior to, or following, LPS or S. aureus stimulation. We found that corneal inflammation was significantly inhibited by Lip-C6 but not control liposomes given prior to, or following, activation by LPS or S. aureus. Furthermore, Lip-C6 did not induce apoptosis of corneal epithelial cells in vitro or in vivo, nor did it inhibit corneal wound healing. Together, these findings demonstrate a novel, anti-inflammatory, nontoxic, therapeutic role for liposomally delivered short-chain ceramide.

Keywords: ceramide, nanoparticles, liposomes, cornea, chemokines

INTRODUCTION

Ceramides are a family of sphingolipids that have well-characterized antiproliferative and proapoptotic activity. Although earlier reports indicate that short-chain ceramides can inhibit activity of human neutrophils [1–4], the putative anti-inflammatory action of short-chain ceramide has not been shown in animal models of inflammation. As neutrophils are a major component of corneal inflammation induced by microbial products, we examined the effect of topical delivery of a liposomal formulation of short-chain ceramide C6 (Lip-C6) in well-characterized mouse models of Staphylococcus aureus and LPS-induced corneal inflammation [5–8].

Bacterial cell wall components such as LPS and killed bacteria can activate the host innate immune response through, and activation of, the TLR family of pathogen recognition molecules [9]. Our previous studies used a murine model of acute corneal inflammation in which synthetic or highly purified TLR2, TLR4, and TLR9 ligands were applied topically to the abraded corneal surface. In this model, chemokine production in the cornea is observed within 6 h, and neutrophil infiltration to the corneal stroma and development of corneal haze peak after 24 h [6, 7]. We also demonstrated that killed S. aureus induces TLR2-dependent corneal inflammation [5], and that TLR-induced activation of corneal epithelial cells and corneal inflammation are dependent on activation of JNK [10]. Our current studies identified an anti-inflammatory role for short-chain ceramide (C6) that inhibits CXC chemokine production by neutrophils and corneal epithelial cells in vitro, and markers of corneal inflammation in vivo, including neutrophil recruitment to the cornea and increased corneal thickness and haze.

Our previous studies also demonstrated that intracellular ceramide accumulation can lead to inhibition of Akt prosurvival pathways and stimulation of caspase activity, resulting in DNA fragmentation and cell death [11, 12]. However, in the current study, inhibition of corneal inflammation by Lip-C6 was observed without appreciable cellular apoptosis or a reduction in wound-healing responses. The current study therefore identifies a previously unknown, inhibitory role for short-chain ceramides in epithelial cell and neutrophil activation in vitro and inhibition of inflammatory responses in vivo.

MATERIALS AND METHODS

Liposome formulation

Liposomes were prepared as described, containing 30 molar percent C6-ceramide, 1,2-disteoroyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy PEG(2000)], and PEG(750)-C6-ceramide [13, 14]. Control liposomes were made up without ceramide but contained the same amount of total lipids. Briefly, lipids were solubilized in chloroform, dried under a stream of nitrogen, and hydrated with a PBS solution at 55°C. The resulting solution was sonicated and underwent extrusion through 100 nm polycarbonate membranes using the Avanti Mini Extruder (Avanti Polar Lipids, Alabaster, AL, USA). Lip-C6 vesicles had an average homogenous size distribution of 80 ± 15 nm, as measured by dynamic light scattering. Lip-C6 and control, ghost liposomes were maintained at 4°C. A National Cancer Institute (Frederick, MD, USA) on-line toxicology and pharmacokinetic analysis noted that the ceramide-incorporated nanoliposome had no significant toxicology in vivo [15].

TLR ligands

Use of S. aureus as a TLR2 ligand in corneal inflammation has been described previously by our group [5, 10]. Briefly, S. aureus strain 8325-4 was provided by Dr. Richard O’Callaghan at the University of Mississippi Medical Center (Jackson, MS, USA). Bacteria were incubated in tryptic soy broth (Difco, Detroit, MI, USA) at 37°C to an OD of 0.3 at 650 nm (~108 CFU/ml), washed three times with PBS (BioWhittaker, Walkersville, MD, USA) and diluted in PBS. A UV Stratalinker (Stratagene, La Jolla, CA, USA), at a setting of 2400 for 15 min was used to inactivate the organisms, and bacterial killing was confirmed by incubating treated bacteria on blood agar plates.

Ultrapure LPS (from Escherichia coli O111:B4) was purchased from Invivo-Gen (San Diego, CA, USA). This reagent, which has no contaminating lipoproteins, reacts specifically with TLR4/myeloid differentiation protein 2 (MD-2).

Human corneal epithelial (HCE) cells

Eyes were obtained from the Cleveland Eye Bank (Cleveland, OH, USA) from individuals who had consented to donate these organs, and all procedures followed the principles articulated in the Declaration of Helsinki. Primary corneal epithelial cells were obtained as described [10, 16]. Briefly, isolated corneas were placed in HBSS containing 10 mg/ml dispase (BD BioSciences, San Diego, CA, USA) and 5 μg/ml gentamicin for 15 h at 4°C. The outer corneal surface was then collected by scraping and treated with 5 mL 0.25% trypsin for 5 min at 37°C. After generating a single-cell suspension, cells were washed and collected by centrifugation and resuspended in keratinocyte serum-free medium (KSFM; Invitrogen-Gibco, Carlsbad, CA, USA) containing epidermal growth factor (EGF) and bovine pituitary extract (BPE). Cells were routinely used at passages 2–5.

The SV-40-immortalized HCE cell line 10.014 pRSV-T (American Type Culture Collection, Manassas, VA, USA), which responds to TLR2 and TLR3, but not TLR4, has been described previously in our laboratory [8, 10]. Briefly, cells were maintained in culture with KSFM, supplemented with BPE and human recombinant EGF (Invitrogen Corp., Carlsbad, CA, USA) at 37°C and 5% CO2. When the cells reached 70–80% confluency, they were placed in KSFM lacking EGF (KSFM-EGF) 24 h prior to TLR stimulation.

In vitro neutrophil activation

The human HL-60 cell line was maintained in RPMI with 10% FBS, and incubated 5 days in 1.2% DMSO to generate the neutrophil phenotype [17]. Cells were placed into 96-well plates at 1 × 105 per well and incubated 6 h with S. aureus or LPS.

To obtain murine neutrophils, mice were injected with 1 ml 9% casein, 16 h and 3 h prior to peritoneal lavage, and cells were layered onto a sterile 90% Percoll gradient (Pharmacia, Biotech, Piscataway, NJ, USA). The neutrophil population was recovered from the second layer on the gradient as determined by cytology and routinely yielded 95–100% pure neutrophils, as described in our previous studies [5, 18, 19]. Neutrophils were incubated in DMEM for 2 h at 37°C with 50 ng/ml GM-CSF and stimulated for 15 h with S. aureus or LPS. Viability was >95% as determined by Trypan blue exclusion.

Chemokine immunoassays

Neutrophil chemokines CXCL1, CXCL5, and CXCL8/IL-8, produced by HCE cells, were measured in culture supernatants by ELISA, according to the manufacturer’s directions (R&D Systems, Minneapolis, MN, USA). The limit of detection for each assay was 5 pg/ml.

SDS-PAGE and Western blot analysis

For immunoblot analysis, equivalent amounts of protein were electrophoresed on denaturing and reducing 8% polyacrylamide gels and transferred to a nitrocellulose membrane, which was blocked by 5% nonfat dry milk and then incubated with antibody specific for total or phospho (P)-JNK and p38MAPK. (Cell Signaling Technology, Beverly, MA, USA). Antibody specific for β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used for immunoblot detection at a dilution of 1:3000. Peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and used at a dilution of 1:5000. Secondary antibody binding was visualized using a commercial chemiluminescence detection kit (Amersham Bioscience, Piscataway, NJ, USA). The intensity of the bands in X-ray film was quantified by “ImageJ” software and calculated as a ratio of P-p38 and P-JNK over total p38 and JNK.

Mouse model of corneal inflammation

C57BL/6 mice (females, 6–8 weeks old) from The Jackson Laboratory (Bar Harbor, ME, USA) were anesthetized by i.p. injection of 0.4 ml 2,2,2-tribro-moethanol, a 1-mm diameter area of the central corneal epithelium was defined using a trephine, and epithelium was abraded using an Algerbrush™ (Richmond Products, Albuquerque, NM, USA). Corneas were then treated with ultrapure LPS from InvivoGen as described [6, 7] or with S. aureus strain 8325 that had been killed by exposure to UV radiation [5]. Lip-Cb treatments are described in Results. Mice were maintained in specific pathogen-free conditions in microisolator cages and were treated 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.

Immunohistochemistry

Neutrophils were identified in the corneal stroma with the anti-murine neutrophil antibody NIMP-R14 using established protocol in our laboratory [5–7, 19]. Briefly, eyes were snap-frozen in liquid nitrogen, and 5 μm sections were prepared on a Micron HM500 M cryostat. Sections were fixed in acetone, blocked with 1% FCS/PBS, and incubated 2 h with NIMP-R14, diluted 1:100 in 1% FCS/TBS (1% FCS/PBS). After washing, corneal sections were incubated with FITC-conjugated rabbit anti-rat antibody (Vector Laboratories, Burlingame, CA, USA), diluted 1:200 in 1% FCS/PBS. Slides were mounted in Vectashield containing 4′,6-diamidino-phenylindole (DAPI; Vector Laboratories), and the number of neutrophils in each section was determined by direct counting. In the absence of primary antibody or using an isotype control, only background staining was detected (not shown).

In vivo confocal microscopy analysis of corneal thickness and haze

In vivo analysis of cellular infiltration was accomplished by in vivo confocal microscopy using a Nidek Confoscan™ (Fremont, CA, USA) as described [6]. Briefly, mice were anesthetized and immobilized, and the cornea was examined using a 40× objective with a transparent gel (Genteal, Novartis Ophthalmics, Duluth, GA, USA) as a medium. A series of images of the entire cornea was captured using Navis™ software, and stromal thickness (area between basal epithelium and corneal endothelium) was measured directly using the associated Navis™ software. To measure stromal haze, the light intensity values of each 1–2 μm image of the corneal stroma was exported into Prism (Graph Pad Software, San Diego, CA, USA), and the total area under the curve was then calculated as described previously [5, 6].

Wound-healing assays

The corneal surface was abraded as described above. At each time-point, mice were anesthetized, and 0.25% fluorescein solution (Bausch and Lomb, Inc., Rochester, NY, USA) was applied as a single drop to the ocular surface. Mice were examined for fluorescein binding by microscopy, corneas were photographed, and the area of the lesion was calculated using ImagePro™ software (MediaCybernetics, Bethesda, MD, USA). Mice were then euthanized, eyes were then fixed in 10% formaldehyde and embedded in paraffin, and 5 μm sections were stained with H&E. Wound healing was then confirmed by examining regeneration of the corneal epithelium.

Apoptosis assays

Corneal epithelial cells or 5 μm frozen corneal sections were incubated with terminal TUNEL reagents according to the manufacturer’s directions (Roche, Penzberg, Germany). TUNEL-positive cells were detected by fluorescence microscopy and quantified by direct counting.

Statistics

Statistical analysis was performed using an unpaired t-test (Prism, Graph Pad Software). A P value of less than 0.05 was considered significant.

RESULTS

Lip-C6 inhibits TLR activation of HCE cells

Previous studies from our laboratory showed that S. aureus stimulates production of neutrophil chemokines CXCL1, CXCL5, and CXCL8 (IL-8) by HCE cells [10, 20, 21]. Furthermore, we showed that TLR ligands stimulate CXC chemokine production in murine corneal epithelium [5–7]. To determine the effect of Lip-C6 on production of these CXC chemokines, HCE cells were exposed briefly to 4–16 μM Lip-C6 or control/ghost (non-C6) liposomes, washed, and incubated with UV-killed S. aureus. As a further control, we used Lip-sphingosine-1-P (S1P), a ceramide metabolite linked to mitogenesis [12]. As shown in Figure 1A, CXCL1, CXCL5, and CXCL8 were produced in response to stimulation with S. aureus. Control (ghost) liposomes had no effect on constitutive or stimulated chemokine production; however, preincubation with 6, 12, or 18 μM Lip-C6 completely inhibited S. aureus-induced CXCL1, CXCL5, and CXCL8 production. In contrast, S1P did not reduce chemokine production, and 16 μM S1P actually augmented CXCL8/IL-8 production by S. aureus.

Fig. 1.

Fig. 1

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Dose-dependent effect of Lip-C6 on CXC chemokine HCE cells. (A) The HCE-T cell line was preincubated 40 min with Lip-C6, Lip-S1P, or control [ghost (Gh)] liposomes, washed, and stimulated with inactivated S. aureus, which activates TLR2 [5]. After 6 h, supernatants were collected, and CXCL1/growth-related oncogene α (GRO-α), CXCL5/epithelial-derived neutrophil-activating factor 78 (ENA-78), and CXCL8/IL-8 were measured by ELISA. (B) Primary HCE cells were isolated from donor corneas and stimulated with the synthetic TLR2 agonist tripalmitoyl-S-glyceryl cysteine (Pam3Cys) in the presence of Lip-C6 or control, ghost liposomes. Note that Lip-C6 inhibits chemokine production at each concentration. Values are mean ± SD for triplicate wells and are representative of three independent experiments. m, Tissue culture medium only.

To ascertain if the effect of Lip-C6 on Pam3Cys (TLR2)-stimulated HCE cells also applies to primary epithelial cells, HCE cells were isolated from donor corneas and incubated with the synthetic TLR2 agonist Pam3Cys in the presence of Lip-C6 or ghost liposomes. As shown in Figure 1B, 4 μM, 8 μM, and 16 μM Lip-C6 ablated CXCL1, CXCL5, and CXCL8 production by primary HCE cells.

To determine the early inhibitory effect of Lip-C6, HCE cells were preincubated with Lip-C6 or control, ghost liposomes prior to addition of S. aureus (5 μM Lip-C6 or control was based on the inhibitory effects found in the previous experiment).

Cell supernatants were collected at 3 h, 6 h, and 12 h, and chemokines were measured by ELISA. As shown in Figure 2, cytokine production by Lip-C6-treated cells was significantly lower than control cells as early as 3 h and continued until 12 h after S. aureus stimulation. Taken together, these data clearly demonstrate that Lip-C6 inhibits TLR2-induced CXC chemokine production by HCE cells in a dose- and time-dependent manner.

Fig. 2.

Fig. 2

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Time-dependent, inhibitory effect of Lip-C6 on CXC chemokine production by HCE cells. (A–C) HCE (HCE-T) cells were preincubated 40 min with 5 μM Lip-C6 or control (ghost) liposomes, washed, and stimulated with S. aureus. After 3 h, 6 h, or 12 h, supernatants were collected, and CXCL1/GRO-α, CXCL5/ENA-78, and CXCL8/IL-8 were measured by ELISA. (D) Primary HCE cells were isolated from donor corneas, preincubated 40 min with Lip-C6 or control liposomes, and stimulated with S. aureus. Supernatants were collected after 3 h, 6 h, or 12 h, and cytokines were measured as before. *, P < 0.05; **, P < 0.01 C6 compared with Gh.

Lip-C6 inhibits S. aureus-induced p38 and JNK phosphorylation in HCE cells

Previous studies from our laboratory and others demonstrated that JNK and p38, proinflammatory and proapoptotic members of the MAPK family, are activated in HCE cells in response to TLR activation, and that JNK is essential for CXC chemokine production [10, 20, 21]. To examine the effect of Lip-C6 on phosphorylation of p38 and JNK, HCE cells were incubated with 5 μM Lip-C6 or ghost liposomes prior to stimulation with S. aureus. HCE cells were then processed for SDS-PAGE and Western blot analysis using antibody to total and phosphorylated JNK and p38.

As shown in Figure 3, JNK and p38 were phosphorylated after stimulation with S. aureus. Furthermore, phosphorylation was blocked by Lip-C6 but not by control (ghost) liposomes. As p38 and JNK are involved in inflammatory processes, these findings indicate that the anti-inflammatory effect of Lip-C6 is partially due to inhibition of p38 and JNK phosphorylation.

Fig. 3.

Fig. 3

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Effect of Lip-C6 on p38 and JNK phosphorylation. HCE cells were incubated with 5 μM Lip-C6 or ghost liposomes for 30 min prior to addition of S. aureus. After 6 h, cells were processed for SDS-PAGE and Western blot analysis using antibodies to phosphorylated and nonphosphorylated forms of p38 and JNK. Bands were scanned by densitometry, and the ratio of phosphorylated to nonphosphorylated forms was quantified, and the mean ± SEM of three independent experiments is shown. Note inhibition of P-p38 and P-JNK after incubation with Lip-C6.

Lip-C6 does not induce apoptosis in corneal epithelial cells

Our previous studies demonstrated a proapoptotic effect of Lip-C6 liposomes on tumor cells, but not untransformed cell lines [13, 14]. Therefore, we next determined if Lip-C6-mediated inhibition of CXC chemokines was a result of induction of a proapoptotic response. HCE cells were incubated with S. aureus and 5 μM Lip-C6 or ghost liposomes for 18 h, and apoptosis was measured by TUNEL assay. As shown in Figure 4, A and B, fewer than 2% TUNEL-positive cells were detected in any of the groups, including Lip-C6-treated cells, whereas 100% cells were TUNEL-positive after DNase treatment. This finding indicates that Lip-C6 inhibits CXC chemokine production without inducing apoptosis.

Fig. 4.

Fig. 4

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Effect of Lip-C6 on HCE cell apoptosis. (A) HCE cells were incubated with DNase I (positive control; a) or with 5 μM Lip-C6 (b), and apoptosis was detected by TUNEL assay. (c and d) Corresponding DAPI-stained cultures, which identify cell nuclei. (B) Two hundred cells were counted, and the percent TUNEL-positive cells is shown. Note that Lip-C6 did not induce apoptosis in these cells.

Lip-C6 inhibits S. aureus- and LPS-induced neutrophil activation

As neutrophils are also critical cells in the pathogenesis of LPS-induced keratitis [7], and human and mouse neutrophils respond to most TLR agonists [5, 22], we next examined the effect of Lip-C6 on S. aureus and LPS-induced neutrophil CXC chemokine production. A human neutrophil cell line (HL-60) was stimulated in vitro with S. aureus or LPS, and CXCL8/IL-8 was measured by ELISA. As shown in Figure 5A and B, CXCL8/IL-8 is produced by the human neutrophil cell line in response to S. aureus or LPS stimulation; however, preincubation with 4–16 μM Lip-C6 significantly inhibits CXC production compared with ghost liposomes.

Fig. 5.

Fig. 5

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The effect of Lip-C6 on S. aureus- and LPS-induced inflammation on human and murine neutrophils. (A and B) The human neutrophil cell line (HL-60) was preincubated with 4, 8, or 16 μM Lip-C6 or ghost liposomes 30 min prior to stimulation with S. aureus (A) or LPS (B). After 6 h, CXCL8/IL-8, in cell-free supernatants, was measured by ELISA. Results are mean ± SD of three replicate wells, and data are representative of three independent experiments. Note that Lip-C6 inhibited CXC chemokine production by TLR2- and TLR4-stimulated human neutrophils compared with ghost liposomes. (C–E) Murine peritoneal neutrophils (>95% pure) were incubated overnight with S. aureus (C and D) or with LPS (E and F) in the presence of Lip-C6 or ghost liposomes. CXCL1/keratinocyte-derived chemokine (KC; C and E) and CXCL2/MIP-2 (D and F) were measured by ELISA. Results are mean ± SD of three replicate wells per sample, and data are representative of three independent experiments. P values show significant differences between ghost and Lip-C6 groups at 15 μM.

As CXC chemokine production by neutrophils is also important in a mouse model of keratitis [19], we also examined the effect of Lip-C6 on murine neutrophils. As shown in Figure 5, C–E, preincubation of a >95% population of murine peritoneal neutrophils with Lip-C6 confirmed our findings with human neutrophils, as 6 μM or 15 μM Lip-C6 inhibited S. aureus (Fig. 5C and D)- and LPS (Fig. 5E, F)-induced CXCL1/KC and CXCL2/MIP-2 production. Note that inhibition of LPS-induced CXCL2 production by 15 μM ghost liposomes was not reproducible (Fig. 5F), in contrast to Lip-C6 versus ghost liposomes for LPS- and S. aureus-induced CXCL1 and CXCL2, which was highly reproducible (Fig. 5, B and C).

Lip-C6 inhibits neutrophil recruitment to the cornea in S. aureus and LPS-induced inflammation

Having demonstrated that Lip-C6 inhibits CXC chemokine production in HCE cells without inducing apoptosis, we next examined the effect of Lip-C6 in vivo. Our previous studies demonstrated that epithelial exposure to S. aureus, bacterial LPS, and other bacterial products to the surface of an abraded cornea induces an inflammatory response that is characterized by neutrophil infiltration and increased corneal thickness and haze, and by elevated expression of cytokines and vascular cell adhesion molecules [6, 7]. To determine if Lip-C6 inhibits LPS- or S. aureus-induced corneal inflammation, mice were treated with 2 nMoles (811 ng) Lip-C6 or control liposomes, administered either by direct injection into the subconjunctival space (in 2 μl); applied topically after inducing a 1-mm diameter corneal abrasion; or applied using both modalities. After 30 min, abraded corneas were treated with S. aureus or LPS. Mice were euthanized 18 h later, 5 μm cornea sections were immunostained using NIMP-R14, and the number of neutrophils per section was determined by direct counting.

As shown in Figure 6, upper panels, neutrophils were absent in naïve corneas, whereas corneas stimulated with S. aureus (in PBS) had a pronounced neutrophil infiltrate. The number of neutrophils in corneas pretreated with control, ghost liposomes was not different from PBS controls; however, corneas pretreated with Lip-C6 had significantly fewer neutrophils than ghost controls, whether administered by subconjunctival injection, topical application, or both. Similar results were found in corneas stimulated with LPS (Fig. 6, lower panels), with Lip-C6 significantly inhibiting neutrophil infiltration regardless of the mode of application. The total Lip-C6 added (2 nMoles; 811 ng) represents the actual amount of lipid applied in 2 μl and was determined from dose-response experiments to determine 50% reduction of neutrophils in the cornea (data not shown); therefore, continuing studies used this amount of Lip-C6 or ghost liposomes.

Fig. 6.

Fig. 6

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The effect of subconjunctival and topical application of Lip-C6 on neutrophil recruitment to the corneal stroma in mouse models of S. aureus- and LPS-induced corneal inflammation. Lip-C6 (2 nMoles; 811 ng) or ghost liposomes were injected into the subconjunctival space, topically applied to a 1-mm diameter region in the central cornea, or were applied by both methods. After 40 min, corneas were stimulated with inactivated S. aureus (upper panels) or LPS (lower panels), and all were given a second topical application 6 h later. After 24 h, eyes were snap-frozen, and neutrophil numbers in a 5-μm corneal section were determined after immunostaining. Data points represent individual corneas from groups of five or six mice. Note that corneas given Lip-C6 by each of these protocols had significantly less neutrophils than control ghost liposomes. The experiment was repeated three times with similar results.

Lip-C6 inhibits S. aureus- and LPS-induced, elevated corneal thickness and haze

To assess the effect of topical Lip-C6 on corneal thickness and haze, corneas were abraded as before, and 100 μM Lip-C6 or ghost liposomes were added 1 h before and 6 h after stimulation with 1 × 107 UV-inactivated S. aureus (Fig. 7, upper panels) or with LPS (Fig. 7, lower panels). After 24 h, neutrophils were examined as before, and corneal thickness and haze were measured by in vivo confocal microscopy (Confoscan™) using Navis™ software as described previously [5, 6] and in Materials and Methods. Results show that Lip-C6 administered topically significantly inhibited neutrophil recruitment to the corneal stroma in this repeat experiment, and also prevented development of increased corneal thickness and haze induced by S. aureus or LPS.

Fig. 7.

Fig. 7

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Effect of topical Lip-C6 on S. aureus- and LPS-induced, increased corneal thickness and haze. A wound (1 mm diameter) was made in the central corneal stroma of C57BL/6 mice, and 2 nMoles (811 ng) topical Lip-C6 was given 1 h before and 6 h after exposure to S. aureus (upper panels) or LPS (lower panels). After 24 h, neutrophils were detected by immunohistochemistry, corneas were by in vivo confocal microscopy, and corneal thickness and haze were calculated as described in Materials and Methods. Data points represent individual corneas from groups of four to eight mice, and the experiment was repeated twice with similar results.

Therapeutic effect of Lip-C6 on corneal inflammation

To determine if Lip-C6 has inhibitory activity if applied after induction of corneal inflammation, corneas were abraded and stimulated with S. aureus and 6 h later, given topical Lip-C6 or liposome controls. Neutrophil recruitment to the corneal stroma was examined 18 h later (24 h after initial induction). As shown in Figure 8, Lip-C6-treated corneas had significantly less neutrophils than control corneas (P<0.05), thereby demonstrating potential therapeutic in addition to preventive activity of Lip-C6 on corneal inflammation.

Fig. 8.

Fig. 8

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Therapeutic (post-induction) effect of Lip-C6 on S. aureus-induced neutrophil recruitment. Corneal inflammation was initiated by abrasion and exposure to S. aureus as before. Lip-C6 or ghost liposome (Gh) control 2 nMoles (811 ng) was added topically after 6 h, and eyes were processed for immunohistochemistry after 24 h. Data points represent individual corneas from five mice per group, and the experiment was repeated twice with similar results.

Lip-C6 does not inhibit corneal epithelial wound healing

Earlier studies from this laboratory and others demonstrated that soluble C6 ceramide has an antiproliferative effect on vascular smooth muscle cells and on breast cancer tumor cells [13, 23–25]. As corneal wound healing requires epithelial cell proliferation and migration, we next examined the effect of Lip-C6 in this process. A 1-mm diameter full-thickness wound was made in the corneal epithelial layer as described above, and corneas were pretreated for 30 min with Lip-C6 or ghost liposomes and exposed to S. aureus. Fluorescein was added to the corneal surface at 0 h, 6 h, and 24 h after abrasion. As fluorescein binds only to sites where the corneal epithelium is not intact or contiguous, fluorescein binding shows the area of the wound, and conversely, fluorescein exclusion indicates wound healing. Figure 9A shows fluorescein staining of the wound at 0 h and 6 h in control (Ghost) and Lip-C6-treated corneas exposed to S. aureus. In contrast, fluorescein was not detected after 24 h, indicating complete wound healing. The rate of corneal wound healing was not inhibited by Lip-C6 or control liposomes in the presence of S. aureus (Fig. 9B). To confirm this observation, we examined H&E-stained sections at these time-points. As shown in Figure 9C, the corneal epithelium of Lip-C6-treated mice, which is absent 6 h after epithelial abrasion, is present after 24 h, indicating that the epithelial layer has regenerated. Similar results were observed in all control groups (data not shown), thereby demonstrating that the anti-inflammatory activity of C6-ceramide is not a result of impaired corneal wound healing.

Fig. 9.

Fig. 9

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Effect of Lip-C6 on corneal epithelial wound healing. A 1-mm diameter corneal epithelial abrasion was made, and corneas were treated with 2 nMoles (811 ng) topical Lip-C6 or ghost liposomes 40 min prior to and 1 and 6 h after exposure to S. aureus. A fluorescein solution (0.25%) was added after 0 h, 6 h, or 24 h to detect the epithelial wound, and the diameter was calculated by image analysis. (A) Representative images of fluorescein binding at indicated time-points. (B) Diameter of wound treated with LipC6 or ghost liposomes (mean of five corneas). Note that there is no inhibitory effect of Lip-C6 on the rate of wound healing. (C) Representative H&E-stained corneal sections 6 h and 24 h after epithelial (Epi) abrasion and exposure to Lip-C6. Note the loss of epithelium at 6 h and regeneration after 24 h; no differences in epithelial regeneration were noted among groups treated with Lip-C6 or controls (data no shown). (D) Corneal sections were also stained with TUNEL reagents (green) to determine the effect on epithelial cell apoptosis, and nuclei were stained with DAPI (blue). Representative sections are shown of naïve corneas (a) or corneal sections after treatment with Lip-C6 and S. aureus (b). TUNEL-positive cells were detected in the stroma and not the epithelium of S. aureus-treated mice. These data are representative of three independent experiments.

To determine if Lip-C6 induces corneal epithelial cell apoptosis, corneal sections were stained for TUNEL reactivity and counterstained with DAPI. Figure 9D shows that TUNEL-positive cells were not detected in the stroma or epithelium of naïve corneas (a) or corneas treated with Lip-C6 and S. aureus (b). TUNEL-positive corneal epithelial cells were not detected in any of the experimental groups (not shown), thereby demonstrating that Lip-C6 has no proapoptotic effect on epithelial cells in vivo.

These observations support the conclusion that Lip-C6 suppresses corneal inflammation by inhibiting CXC chemokine production and resultant neutrophil recruitment to the corneal stroma rather than promoting epithelial cell apoptosis.

DISCUSSION

Results of our in vitro and in vivo studies support a novel, anti-inflammatory role for Lip-C6. Despite the documented role of ceramide in promoting apoptosis in several cell types [26–28], this does not appear to be the mechanism of action in LPS and S. aureus-induced corneal inflammation. Rather, Lip-C6 inhibits proinflammatory p38 and JNK phosphorylation in addition to CXC chemokine production in HCE cells, and inhibits chemokine production in human and murine neutrophils. These anti-inflammatory effects of Lip-C6 on cells that mediate corneal inflammation are consistent with the effect of local (topical) Lip-C6 administration in vivo, which is to inhibit neutrophil infiltration to the corneal stroma and development of corneal haze.

Our studies are the first definitive observation that short-chain ceramide formulations exert anti-inflammatory actions in vivo, and are consistent with previous reports showing an inhibitory (anti-inflammatory) role for ceramide on the respiratory burst in TNF-α-activated human neutrophils [1] and on IgG-dependent neutrophil phagocytosis [2]. Short-chain ceramide also limits oxidant H2O2 release from fMLP-adherent human neutrophils [3] and inhibits superoxide formation and calcium influx [4]. In addition, ceramide can inhibit LPS/ TLR4-induced IL-8 synthesis in endothelial cells [29], and C8 ceramide down-regulates LPS-stimulated mast cell cytokine production by inhibiting the PI-3K-Akt pathway [30]. These findings are supported by results from the current study in which TLR-induced cytokine production is inhibited, and imply that C6 ceramide has a similar inhibitory effect on neutrophils in the corneal stroma. It is also possible that stromal fibroblasts contribute to the inflammatory response and are inhibited by Lip-C6; however, our data show clearly that short-chain ceramide in liposome formulation limits keratitis by reducing activation and production of CXC chemokines by corneal epithelial cells and thereby limits recruitment of neutrophils to the corneal stroma. Although our future studies will examine more completely the mechanism of action of C6 on corneal inflammation, results presented here indicate that CXC secretion by HCE cells involves Lip-C6-inhibiting, proinflammatory cascades that involve p38 and JNK. This is consistent with our recent findings that TLR-induced activation of these cells is dependent on JNK phosphorylation [10]. In addition to corneal epithelial cells, neutrophil activation is inhibited by Lip-C6, which represents a second target of anti-inflammatory activity. Taken together, our studies support low-dose, nanoliposome-formulated, short-chain ceramides as a potential therapy to reduce corneal thickening or haze via inhibition of TLR proinflammatory signaling.

Formulating short-chain ceramides in nanoscale (<100 nm)-pegylated liposomes (30 molar percent bioactive ceramide) has significant advantages as a potential therapeutic. These ceramide nanoliposomes provide a useful, topical delivery modality for corneal disease. In addition, the Nanotechnology Characterization Laboratory of the National Cancer Institute examined the toxicology and pharmacokinetic analysis of the ceramide-incorporated nanoliposome and noted an absence of significant, toxicological effects in vivo [15]. Short-chain ceramides as a component of pegylated liposomes are protected from premature metabolism and exert their biological activity via intrabilayer movement [31]. As shown in models of atherosclerosis and diabetes and confirmed in the present study, it is often the phosphorylated or glycosylated ceramide metabolites and not ceramide itself that mediate the proinflammatory actions of sphingolipids [32, 33]. Thus, the use of short-chain ceramides, possibly protected within nanotechnology-based formulations, can mitigate proinflammatory cascades in vivo. Furthermore, pre- and post-treatment anti-inflammatory actions of ceramide nanoliposomes demonstrated here in models of corneal inflammation suggest that short-chain ceramide formulation would also have broader effectiveness by inhibiting other clinical manifestations of inflammation. Future studies will examine this possibility.

Acknowledgments

This research was funded by National Institutes of Health Grants RO1EY14362 and P30EY11373 (E. P.), a sponsored research agreement with Bausch and Lomb (E. P.), and by NIH grant EY015800 (M. K. and T. F.). Additional support was obtained from The Research to Prevent Blindness Foundation and the Ohio Lions Eye Research Foundation. E. P. is a recipient of a Research to Prevent Blindness Senior Investigator Award. M. K. has licensed similar nanotechnologies to Tracon Pharmaceuticals for unrelated applications. M. K. is also Chief Medical Officer for KeystoneNano, Inc. to develop unrelated nanotechnology delivery systems. We thank Dr. Scott Howell for technical assistance with this study.

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