Galectin-1-Mediated Suppression of Pseudomonas aeruginosa-Induced Corneal Immunopathology

Abstract

Corneal infection with Pseudomonas aeruginosa leads to a severe immunoinflammatory lesion often causing vision impairment and blindness. Although past studies have indicated a critical role for CD4⁺ T cells, particularly Th1 cells, in corneal immunopathology, the relative contribution of recently discovered Th17 and Treg cell is undefined. In this study, we demonstrate that after corneal P. aeruginosa infection, both Th1 and Th17 cells infiltrate the cornea with increased representation of Th17 cells. In addition to Th1 and Th17 cells, Treg also migrate into the cornea during early as well as late stages of corneal pathology. Moreover, using galectin-1 (Gal-1), an immunomodulatory carbohydrate-binding molecule, we investigated whether shifting the balance among various CD4⁺ T cell subsets can modulate P. aeruginosa-induced corneal immunopathology. We demonstrate here that local recombinant Gal-1 (rGal-1) treatment by subconjunctival injections significantly diminishes P. aeruginosa-mediated corneal inflammation through multiple mechanisms. Specifically, in our study, rGal-1 treatment significantly diminished corneal infiltration of total CD45⁺ T cells, neutrophils and CD4⁺ T cells. Furthermore, rGal-1 treatment significantly reduced proinflammatory Th17 cell response in the cornea as well as local draining lymph nodes (DLN). Also, rGal-1 therapy promoted antiinflammatory Th2 and IL-10 response in secondary lymphoid organs. Collectively, our results indicate that corneal P. aeruginosa infection induces a strong Th17-mediated corneal pathology and treatment with endogenously derived protein such as Gal-1 may be of therapeutic value for the management of bacterial keratitis, a prevalent cause of vision loss and blindness in humans worldwide.

Introduction

Corneal infection with Pseudomonas aeruginosa is a leading cause of infectious microbial keratitis and associated vision impairment in humans worldwide (1–3). This bacterial keratitis is characterized by rapid inflammatory response with suppurative coagulative necrosis often irreversibly damaging corneal tissue architecture, leading to vision loss (4). Widespread use of contact lenses and the ability of P. aeruginosa to grow in nutrient deprived conditions such as various ophthalmic solutions poses a serious threat of vision impairment due to P. aeruginosa keratitis. (5–7). Currently, bacterial keratitis is mainly controlled by therapeutic topical administration of antibiotics (8). However, constant emergence of antibiotic resistant bacteria poses a serious challenge for effective management of microbial keratitis (9, 10). Therefore, understanding the pathogenesis of P. aeruginosa corneal infection, particularly the different factors and cells that either contribute to or inhibit the corneal pathology, would reveal significant information to design novel therapeutics.

Past studies using a mouse model of P. aeruginosa-induced bacterial keratitis have shown the complex interplay of numerous factors secreted from both host as well as pathogen (4, 11). The inflammatory response in the cornea is mainly initiated and maintained by sequential migration and activation of proinflammatory cells from both innate and adaptive immune response against invading corneal P. aeruginosa infection (4). The role of CD4⁺ effector T cells, particularly CD4⁺ IFN-γ⁺ T (Th1) cells, is well documented in the pathogenesis of P. aeruginosa infection of the cornea (12–14). In this respect, it is known that mice strains such as C57BL/6, which are biased toward a strong Th1 response, are susceptible to a severe form of pathology characterized by corneal perforation (13). In contrast, BALB/C mouse strain that dominantly induces Th2 (CD4⁺ IL-4⁺ T cells) response is more resistant to corneal perforation (13). Furthermore, studies have shown that shifting the balance from a Th1 to Th2 response in C57BL/6 mice significantly diminishes pathology and protects mice from corneal perforation after P. aeruginosa infection (15, 16). Although these reports indicate an obvious involvement of Th1 cells as a principal orchestrator of corneal immunopathology after P. aeruginosa infection, the actual tissue damage to the corneal epithelial and stromal tissue layers is primarily mediated by uncontrolled migration and activation of neutrophils, and their secretion of various proinflammatory molecules in the cornea (4, 11, 17, 18).

Little is known about the role of recently discovered Th17 cells (CD4⁺ IL-17A⁺ T cells) in the immunopathology of P. aeruginosa keratitis. Th17 cells, a new subset of CD4⁺ T cells that produce IL-17A, have been shown to play an important role in various autoimmune and immunopathological disorders by enhancing granulopoiesis as well as neutrophil activity at the site of inflammation (19). IL-17A is a proinflammatory cytokine that contributes to the local inflammatory response through increased production of various chemokines and cytokines essential for migration and activation of neutrophils at the site of inflammation (20–22). Since the neutrophil is the predominant cell type that contributes to corneal tissue damage post P. aeruginosa infection, it is plausible that Th17 cells and resulting IL-17A are key factors responsible for the severe tissue destruction in P. aeruginosa keratitis. In support of this notion, Zaidi et al. (23) have shown that topical neutralization of IL-17A during P. aeruginosa corneal infection reduces neutrophil influx and pathology.

Recent discovery of CD4⁺ Foxp3⁺ regulatory T cells (Treg) and their critical role in the suppression of the effector T cell response represents a promising therapeutic strategy to control various autoimmune and chronic immunopathological disorders (24). Accordingly, recent studies have demonstrated that various strategies that promote increased representation of Treg over Th1 and/or Th17 can be beneficial to host in various autoimmune and chronic pathological disorders (24–27). Moreover, host produces antiinflammatory molecules which curtail proinflammatory Th1 and Th17 response and promote antiinflammatory Treg and Th2 response (28–30). In this regard, galectin-1 (Gal-1), a member of the galectin family of mammalian β-galactoside-binding proteins, has been shown to play an immunomodulatory role during various immunoregulatory processes (28, 31–33). Gal-1 exerts its antiinflammatory effect through a variety of mechanisms including induction and differentiation of Treg, suppression of Th1 and Th17 differentiation, increased apoptosis of activated Th1 and Th17 cells, increased production of antiinflammatory cytokines and induction of CD4⁺ IL-10⁺ T cells (Tr1) (28, 34–41). Accordingly, Gal-1 has been shown to diminish Th1- and Th17-mediated immunopathology through increased representation of Treg/Tr1/Th2 cells over Th1 and Th17 responses (28, 36, 40, 42, 43). However, the role of Treg and modulation of different CD4⁺ T cell subsets during corneal P. aeruginosa-induced immunopathology is poorly understood.

In the present report, we demonstrate for the first time that both Th1 and Th17 cells infiltrate the cornea after P. aeruginosa infection. Moreover, we show that Th17 cells are far more numerous than Th1 cells during the late stage of corneal immunopathology. Additionally, Treg cells also migrate into the cornea during the early as well as the late stages of bacterial keratitis. Moreover, we show that application of recombinant Gal-1 (rGal-1) by subconjunctival injections after ocular P. aeruginosa infection significantly diminishes corneal lesion severity through modulation of Th17 and Treg responses. Interestingly, rGal-1 treatment shifted the proinflammatory Th17-mediated pathology towards antiinflammatory Th2 and Tr1 type immune responses.

Material and Methods

Mice and bacterial strains

C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals were housed in animal facilities approved by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) at Tufts University and all experimental procedures were in complete agreement with the Association for Research in Vision and Ophthalmology resolution on the use of animals in research. A P. aeruginosa cytotoxic strain 6077 was used for corneal infection. Different doses from 2 × 10⁵ to 5 × 10⁵ CFU/eye were evaluated for severity of corneal pathology. For kinetics experiments, a dose of 3.5 × 10⁵ CFU/eye was used to infect mice, whereas for rGal-1 treatment experiments, a slightly lower dose of 2.5 × 10⁵ CFU/eye was used to infect mice. Bacterial strain was grown overnight on Tryptic Soy Broth agar plates at 37°C before each infection.

Corneal Infection

Corneal infections of mice were conducted under deep anesthesia induced by i.p. injection of ketamine and xylazine. Central corneas of mice were scarified with three parallel 1 mm incisions using a 26-gauge needle, and a 5-ul drop containing the appropriate dose of bacteria was applied to the eye.

Clinical Scoring

The eyes were examined on different days postinfection (pi) for the development of corneal opacity. The following scoring system, graded from 0 to 4, was used as previously described (44). 0- eye macroscopically identical to the uninfected control eye, 1- partial corneal opacity covering the pupil, 2- dense corneal opacity covering the pupil, 3- dense opacity covering the entire anterior segment, 4- perforation of the cornea, phthisis bulbi (shrinkage of the globe post inflammatory disease).

Reagents and Antibodies

CD4-APC (RM4-5), IL-17-PE (TC11-18H10), IFN-γ-FITC (XMG1.2), CD45-PerCp (30-F11), CD45-APC (30-F11), CD11b-PerCp (M170), Ly6G-PE (1A8), anti-CD3 (145.2C11), anti-CD28 (37,51), and GolgiStop (Brefeldin A) were purchased from BD Bioscience (San Jose, CA). Foxp3-PE, and ELISA kit for IL-4, IL-10 and IL-17A were purchased from eBioscience (San Diego, CA). The galectin-1 EST clone expressing human rGal-1 (pQE-60/hGal-1) was kindly provided by Dr. Fu-Tong Liu, Department of Dermatology, University of California, Davis, School of Medicine, Sacramento, CA. The rGal-1 was purified by culturing transformed Escherichia coli with pQE-60/hGal-1 in LB containing ampicillin (50 µg/ml). The production of rGal-1 was induced by adding 1mM isopropyl-β-D-thiogalactoside (IPTG) during last 4 hrs of bacterial culture. Recombinant Gal-1 was purified from bacterial cell lysate by affinity chromatography using lactosyl-Sepharose beads. Briefly, bacterial cell lysate supernatants were incubated with 2 ml of bead volume at 4°C for 1 hr followed by loading of beads on column. The column was washed with 30 ml of 20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.2 mM PMSF, 4 mM β-mercaptoethanol followed by a second washing with 10 ml of wash buffer (PBS containing 0.2 mMPMSF, 4 mM β-mercaptoethanol). Fractions containing rGal-1 were eluted with wash buffer containing 0.1 M lactose and dialyzed against PBS containing 2% glycerol, 4 mM β-mercaptoethanol and stored at −80°C.

rGal-1 Treatment

Subconjunctival injections of rGal-1 in P. aeruginosa infected mice were performed as described previously (45). Briefly, a 2-cm needle attached to a 32-gauge syringe (Hamilton, Reno, NV) was used to penetrate the perivascular region of the conjunctiva, and rGal-1 (20 µg in 10 µl volume) was delivered into the subconjunctival space. P. aeruginosa infected mice were scored for corneal opacity on day 1 pi and animals with similar corneal opacity were randomly divided into two groups. One group of mice was treated with rGal-1 by local subconjunctival injections every alternate day starting from day 1 until day 11 pi. Control mice were injected with 10 µl of vehicle (PBS containing 2% glycerol, 4 mM β-mercaptoethanol).

Detection of corneal infiltrating cells and flow cytometry

P. aeruginosa infected corneas were harvested from different groups of mice at indicated time points pi. Six to eight corneas per group were excised, pooled group wise, and digested with 60 U/ml Liberase (Liberase TL, Roche Diagnostics, Indianapolis, IN) for 35 minutes at 37°C in a humidified atmosphere of 5% CO₂. After incubation, the corneas were disrupted by grinding with a syringe plunger on a cell strainer and single cell suspensions were made in complete RPMI 1640 medium. The single cell suspensions obtained from corneas, draining lymph nodes (DLN) and spleen were stained for different cell surface molecules using conjugated antibodies as described below. Briefly, cells were blocked with an unconjugated anti-CD32/CD16 mAb for 30 min in FACS buffer (2% Fetal Bovine Serum in PBS), and were then incubated with different cocktails of antibodies depending on cell type for 30 min on ice. For CD4⁺ T cells, CD4-APC (RM4-5) and CD45-PerCP (30-F11) were used. For neutrophils, CD45-APC (30-F11), CD11b-PerCP (M170), Ly6G-PE (1A8) mAbs were used. The cell samples were washed three times using FACS buffer followed by fixation in 1% paraformaldehyde.

To analyze the number of IFN-γ, IL-17 and IL-4 producing CD4⁺ T cells, intracellular cytokine staining was performed as previously described with minor modifications (46). Briefly, one million cells from DLN and spleen or total corneal cells were left untreated or stimulated with anti-CD3 (1µg/ml) and anti-CD28 (0.5 µg/ml) for 5 hrs in the presence of GolgiStop at 37°C in 5% CO₂. At the end of the stimulation period, cell surface staining for CD4 was performed as described above. This was followed by cell permeabilization and intracellular cytokine staining using Cytofix/Cytoperm kit (BD Biosciences) in accordance with the manufacturer’s recommendations. For intracellular staining, PE labeled IL-4, FITC labeled IFN-γ and PE labeled IL-17 antibodies were used. To enumerate Foxp3⁺CD4⁺ T cells, cell surface staining was performed for CD4 (APC) followed by intranuclear staining using the fixation/permeabilization kit and an anti-Foxp3-PE mAb (eBioscience) as per manufacturer’s recommendations. Samples were acquired with a FACS Calibur (BD Biosciences) and the data were analyzed using FlowJo software (Tree Star, Ashland, OR).

ELISA

The pooled corneal samples were homogenized using a tissue homogenizer and supernatant was used for analysis. For DLN and spleen, single cell suspensions were collected on day 12 pi. Aliquots of single cell suspensions containing 1 million cells were stimulated with anti-CD3 (2 µg/ml) and anti-CD28 (1 µg/ml) for 48 h at 37°C. The concentrations of IL-4, IL-10 and IL-17A were measured in supernatants by using sandwich ELISA kits (eBioscience) as per manufacturer’s instructions.

Statistical analysis

Unpaired two-tailed Student’s t test was performed to determine statistical significance for disease severity between different groups. P values ≤ 0.05 were considered significant. All experiments were repeated at least two times and results are expressed as means ±SEM.

Results

Corneal P. aeruginosa infection induces a dominant Th17 cell response in the cornea

Past studies have shown that after corneal P. aeruginosa infection, CD4⁺ T cell infiltration in the cornea becomes readily apparent as early as day 3 pi with subsequent migration of activated CD4⁺ T cells by day 5 pi (4, 12–14). Moreover, it has been noted that Th1 cells contribute to corneal pathology and perforation (4, 12–14). Although these studies implied the critical role of IFN-γ producing Th1 cells in P. aeruginosa-induced corneal immunopathology, the relative contribution of recently discovered Th17 cells is not defined. To evaluate the role of different CD4⁺ T cell subsets in P. aeruginosa-mediated corneal pathology, corneas of mice were infected with P. aeruginosa and the severity of corneal opacity and CD4⁺ T cell infiltration was measured at various days pi (Fig. 1). As shown in Fig. 1A–B, P. aeruginosa infected mice showed corneal opacity on day 1 pi with increasing severity as infection progressed. The peak severity of keratitis was noted on day 7 pi (Fig. 1A–B). To characterize the role and relative contribution of different CD4⁺ T cell subsets, mice were sacrificed on day 5 and 8 pi and single cell suspensions were prepared from collagen-digested pooled corneal samples for cellular analysis by flow cytometry. As shown in Fig. 1C, CD4⁺ T cells were evident by day 5 pi with continued infiltration in the cornea on day 8 pi as the disease progressed to a more severe form. Interestingly, intracellular cytokine staining revealed the infiltration of both Th1 and Th17 cells in the cornea on day 5 and day 8 pi (Fig. 1D and E). However, as the disease progressed to a more severe form, the Th17 cells became far more prominent than Th1 cells (Fig 1F). Taken together, these results demonstrate that both Th1 and Th17 cells infiltrate the cornea early after P. aeruginosa infection and that there is an increased representation of Th17 cells with the more severe P. aeruginosa keratitis during the late phase of corneal P. aeruginosa infection.

FIGURE 1. Ocular P. aeruginosa infection induces a strong Th17 cell response in the cornea.

C57BL/6 mice corneas were scarified and infected with 3.5 × 10⁵ CFU of P. aeruginosa. The progression of bacterial keratitis was monitored by measuring corneal opacity scores on day 1, 3, 5 and 7 pi followed by flow cytometric analysis of CD4⁺ T cells infiltrating the cornea on day 5 and 8 pi. (A) Corneal opacity scores on day 0 (uninfected) and day 1, 3, 5 and 7 pi. (B) Representative eye images showing the disease severity at various days pi. (C) Representative FACS plots (left) and cell numbers (right) for CD4⁺ T cells gated on CD45⁺ cells obtained from pooled corneal samples on day 5 and 8 pi. (D–E) Representative FACS plots (left) and cell numbers (right) for IFN-γ (D) or IL-17A (E) secreting CD4⁺ T cells from pooled corneas stimulated with anti-CD3/anti-CD28 for 5 hrs from day 5 and day 8 pi. (F) Total number of Th1 and Th17 cells on day 8 pi. Data are representative of two independent experiments and show mean values ± SEM (combined results of two separate experiments are shown, 5 to 6 corneas/group were used in each experiment). *p≤0.05. Statistical levels of significance were analyzed by the Student t test (unpaired).

Ocular P. aeruginosa infection induces a strong Treg response in the cornea

Past studies have shown that Treg control both Th1- and Th17-mediated cellular response and suppress various autoimmune and immunopathological disorders associated with excessive effector T cell responses (24). In the cornea, primary herpes simplex virus (HSV) infection results in expansion of the Treg population and increasing the representation of Treg over Teffectors diminishes corneal immunopathology (25–27, 47). To study the role of Treg during corneal P. aeruginosa infection, we analyzed corneal infiltration of Treg at various days pi. As shown in Fig. 2A–B, Treg cell infiltration in the cornea was far greater than T effectors (both Th1 and Th17, see Fig. 1D–E) during both early and late stages of P. aeruginosa infection (Day 5: Th1, 7.8 %; Th17, 9.8%; Treg 65%; Day 8: Th1, 5.14%; Th17, 18.1%; Treg, 38.8%). When the ratio of total number of Treg to Teffector cells was compared during early and late stage of P. aeruginosa infection, there was no change in the total number of Treg cells per Th1 cell (Fig. 2C) or Treg cells per Th17 cell (Fig. 2D). Collectively, these results indicate that corneal P. aeruginosa infection induces a steady Treg cell response in the cornea. However, the total number of Th17 cells is significantly increased during the late stage of corneal P. aeruginosa infection indicating that increased representation of Th17 cells could be responsible for a more severe form of corneal pathology.

FIGURE 2. Treg response in the cornea post P. aeruginosa infection.

C57BL/6 mice corneas were scarified and infected with 3.5 × 10⁵ CFU of P. aeruginosa. Flow cytometric analysis of Foxp3⁺ CD4⁺ T cells infiltrating the cornea on day 5 and 8 pi. (A) Representative FACS plots for Foxp3⁺ CD4⁺ T cells obtained from pooled corneal samples on day 5 (left) and 8 (right) pi. (B) total cell numbers for Foxp3⁺ CD4⁺ T cells in P. aeruginosa infected corneas on day 5 and 8 pi. (C) Cell ratios for total numbers of Treg per Th1 cell (C) or Treg per Th17 cell (D). Combined results of two separate experiments are shown as mean values ± SEM, 5 to 6 corneas/group were used in each experiment. *p≤0.05. Statistical levels of significance were analyzed by the Student t test (unpaired).

Treg and Teffector response in lymphoid organs post corneal P. aeruginosa infection

Next, we examined the kinetics of Teffector and Treg responses in local DLN and spleen at various days pi. The results in Fig. 3 show the frequency and total numbers of Th17 and Treg cell populations in DLN and spleen of P. aeruginosa infected mice at different days pi (day 5 and 8 pi) as compared to uninfected control mice. As shown in Fig. 3A–D, there was a significant increase in the frequency as well as the total cell number of Th17 cells in DLN and spleen of P. aeruginosa infected mice. Consistent with the observations made in the cornea, the frequency as well as total number of Th17 cells was significantly higher on day 8 as compared to day 5 pi in DLNs. These data suggest that corneal P. aeruginosa infection induces a strong Th17 dominant immune response in the local DLN (Fig. 3A–B). In contrast, when the frequency and total cell number for Th1 cells were compared, we observed a significant increase only in spleen of P. aeruginosa infected mice as compared to uninfected mice (data not shown).

FIGURE 3. Corneal P. aeruginosa infection induces Th17 and Treg cell response in lymphoid organs.

C57BL/6 mice corneas were scarified and infected with 3.5 × 10⁵ CFU of P. aeruginosa. Flow cytometry was used to detect IL-17A production by CD4⁺ T cells (anti-CD3/anti-CD28 stimulated for 5 hrs) obtained from DLN and spleen of uninfected (day 0), and animals from day 5 and day 8 pi. The frequencies (A) and total cell numbers (B) of IL-17A producing CD4⁺ T cells in DLN of uninfected (day 0) and P. aeruginosa infected (day 5 and day 8 pi) mice are shown. The frequenceis (C) and total cell numbers (D) of IL-17A producing CD4⁺ T cells in spleen of uninfected (day 0) and P. aeruginosa infected (day 5 and day 8 pi) mice are shown. Flow cytometry was used to detect Foxp3 expressing CD4⁺ T cells obtained from DLN and spleen of uninfected (day 0), day 5 and day 8 pi. The total cell numbers of Foxp3⁺ CD4+ T cells in DLN (E) and spleen (F) of uninfected (day 0) and P. aeruginosa infected (day 5 and day 8 pi) mice are shown. Cell ratios for total numbers of Treg per Th17 cell in DLN (G) and spleen (H) of uninfected (day 0) and P. aeruginosa infected (day 5 and day 8 pi) mice are shown. Data are representative of two independent experiments and show mean values ± SEM (n = 8 mice at each indicated time point p.i.). ***p ≤ 0.001,**p ≤ 0.01,*p ≤ 0.05, ns- non-significant. Statistical levels of significance were analyzed by one-way ANOVA test with Tukey’s multiple comparaion test.

As shown in Fig. 3E–F, there was no significant difference in the total number of Treg cells in DLN and spleen from uninfected and P. aeruginosa infected mice at various days pi. However, when the ratio of total number of Treg to Th17 cells was compared between uninfected and P. aeruginosa infected mice, a significant decrease in the proportion of Treg per Th17 cell was noted in both DLN and spleen at day 5 and 8 pi (Fig. 3G–H). In contrast to Th17 cells, there was no significant change in the number of Treg cells per Th1 cell in DLN as well as spleen of corneal P. aeruginosa infected mice as compared to uninfected mice (data not shown). Taken together, these results indicate that local corneal P. aeruginosa infection results in the expansion of Th17 cell response in DLN and spleen. Our data also suggest that a significant reduction in the total number of Treg cells per Th17 cell in DLN and spleen during later stage of keratitis may induce a strong Th17 cell-mediated immunopathology in the cornea.

Gal-1-Mediated Suppression of P. aeruginosa keratitis

Past reports have shown that the severity of CD4⁺ T cell-mediated immunopathological disorders could be influenced by shifting the balance between effector T cells and Treg (25, 27, 42). More recently, various in vivo studies using mouse models have demonstrated that Gal-1, a member of β-galactoside-binding lectin family, suppresses CD4⁺ T effector cell-mediated different autoimmune and immunopathological disorders such as HSV-induced corneal stromal keratitis (36), experimental autoimmune encephalomyelitis (EAE) (48), autoimmune diabetes (49), collagen-induced arthritis (50) and experimental colitis (51). To evaluate whether the administration of rGal-1 after corneal P. aeruginosa infection could influence the balance between Th17 cells and Treg and limit the severity of corneal immunopathology, corneas of C57BL/6 mice were infected with P. aeruginosa and rGal-1 (20 µg in 10 µl per eye) was administered by subconjunctival injections every other day beginning day 1 pi (Fig. 4A). The severity of corneal pathology was recorded every alternate day and compared to the vehicle treated control mice (Fig. 4B). All infected mice on day 1 pi developed corneal opacity to a similar extent. However, as infection progressed, the animals in the rGal-1 treated group showed significantly diminished corneal pathology as compared to control mice (Fig. 4B–D). Interestingly, continued rGal-1 treatment during the course of P. aeruginosa infection not only inhibited the further progression of the corneal lesion but also reversed the increased corneal opacity and corneal pathology to a normal clear cornea (Fig. 4D). Taken together, our results demonstrate that administration of rGal-1 treatment after corneal P. aeruginosa infection significantly reduces the corneal pathology.

FIGURE 4. Administration of rGal-1 after corneal P. aeruginosa infection reduces the severity of bacterial keratitis.

(A), C57BL/6 mice infected with 3.5 × 10⁵ CFU of P. aeruginosa were treated with rGal-1 (20 µg in 10 µl/eye) or control vehicle by local subconjunctival injections from day 1 to 11 pi. The progression of bacterial keratitis was monitored by measuring corneal opacity scores on day 1, 3, 5, 7, 9 and 11 pi. (B) The progression of bacterial keratits was significantly diminished in rGal-1 treated animals compared to control mice treated with vehicle alone. Kinetic analysis for mean scores at each indicated day pi is shown. Data are representative of three independent experiments and show mean values ± SEM (n = 22–25 mice/group; ***p=0.0002,**p= 0.001,*p=0.0117). Statistical levels of significance were analyzed by the Student t test (unpaired). (C) Individual eye scores for corneal opacity on day 11 pi from rGal-1 treated and control group are shown. (D) Representative eye photos on day 1, 3, 5, 7, 9 and 11 pi show corneal lesion severity from rGal-1 treated animals as compared to control group mice. All eye pictures at different days pi are from the same eye of each group.

rGal-1 treatment diminishes infiltration of various immune cell types involved in pathology

To further characterize the mechanisms of the inhibitory effect of rGal-1 treatment on P. aeruginosa-induced bacterial keratitis, the corneal immune cell composition of both control and rGal-1 treated groups was analyzed at day 12 pi by flow cytometry. As shown in Fig. 5A–E, the frequencies and total numbers of CD45⁺ cells, CD4⁺ T cells and neutrophils (CD45⁺ CD11b⁺ Ly6G⁺) were significantly reduced in the corneas of rGal-1 treated group when compared to the vehicle treated control animals. These results show that rGal-1 treatment after corneal P. aeruginosa infection significantly reduces the corneal infiltration of total leukocytes, CD4⁺ T cells and neutrophils.

FIGURE 5. rGal-1 treatment diminishes cellular infiltration in P. aeruginosa infected corneas.

C57BL/6 mice infected with 3.5 × 10⁵ CFU of P. aeruginosa were treated with rGal-1 (20 µg in 10 µl/eye) or control vehicle (PBS) by local subconjunctival injection from day 1 to 11 pi. Representative FACS plots of Ly6G⁺ CD11b⁺ polymorophonuclear neutrophils (A) and CD4⁺ T cells (B) gated on total CD45⁺ cells infiltrated in the corneas of rGal-1 treated (right) or control group animals are shown. Numbers of total CD45⁺ cell, neutrophils, and CD4⁺ T cells gated on CD45⁺ cells infiltrated per cornea of control and rGal-1 treated group are shown in panels C, D and E, respectively. (n = 3, each sample is representative of 5 to 10 corneas). **p<0.01,*p<0.05. Statistical levels of significance were analyzed by the Student t test (unpaired).

rGal-1 treatment diminishes the proinflammatory Th17 cell response in the cornea and lymphoid organs

To assess whether Gal-1 suppresses P. aeruginosa-induced keratitis through a shift in the balance between Teffectors and Treg, CD4⁺ T cell subset composition of infected corneas of rGal-1 treated and control animals was analyzed by FACS at day 12 pi. As shown in Fig. 6A–B, the frequency and total number of Th17 cells (Fig. 6B) were reduced in the corneas of animals treated with rGal-1 compared to the control animals. In contrast, there was no difference in the frequency of Treg population in rGal-1 treated group as compared to control animals (Fig. 6C). Also, when the ratio of total number of Treg cells per Th17 cells was compared between rGal-1 treated and control groups, there was no change in the total number of Treg cells per Th17 cell in rGal-1 treated group (Fig. 6D). In another set of experiments of the same design, the corneas were pooled groupwise and processed to analyze the levels of IL-17A by ELISA. As shown in Fig. 6E, the levels of proinflammatory cytokine IL-17A were significantly diminished in rGal-1 treated group as opposed to vehicle treated control group. In contrast, there was no difference in the levels of the antiinflammatory cytokine IL-10 between the rGal-1 treated and control group (Fig. 6F). Taken together, these data indicate that rGal-1 treatment modulates the severity of P. aeruginosa-induced corneal pathology by reducing the magnitude of proinflammatory Th17 response.

FIGURE 6. rGal-1 treatment diminishes Th17 response in P. aeruginosa infected corneas.

C57BL/6 mice infected with 3.5 × 10⁵ CFU of P. aeruginosa were treated with rGal-1 or control vehicle by local subconjunctival injections from day 1 to 11 pi. Representative FACS plots (A) and total cell numbers (B) for IL-17A secreting CD4⁺ T cells from day 12 pi pooled corneas stimulated with anti-CD3/anti-CD28 for 5 hrs of control (left) and rGal-1 treated (right) animal groups. Representative FACS plots (C) for Foxp3⁺ CD4⁺ T cells from day 12 pi pooled corneas of control (left) and rGal-1 treated (right) mice group. (D) Cell ratios for total numbers of Treg per Th17 cell. IL-17A (E) and IL-10 (F) protein levels per cornea from control and rGal-1 treated group. Data are representative of three independent experiments and show mean values ± SEM (n = 3, each sample is representative of 5 to 10 corneas). *p<0.05. Statistical levels of significance were analyzed by the Student t test (unpaired).

Additionally, studies were performed to quantify the relative composition of Th17 and Treg cells in DLN and spleens from the same mice as those used for corneal studies. As shown in Fig. 7A–D, rGal-1 treatment reduced the percentages of Th17 cells in both DLN and spleen as compared to the control group but did not significantly influence the percentage of Treg populations. Also, there was a significant reduction in total CD4⁺ T cells and Th17 cell numbers in DLN of rGal-1 treated mice as compared to control group animals (Fig. 7E). Similarly, total cell number of CD4⁺ T cells and Th17 cells were reduced in spleen of rGal-1 treated animals (Fig. 7F). Although there was significant reduction in total Treg cell numbers in rGal-1 treated group (Fig. 7E–F), there was no change in the total number of Treg cells per Th17 cell in both DLN and spleen of rGal-1 treated and control mice (Fig. 7G–H). Furthermore, when the cytokine levels of IL-17A and IL-10 from cell supernatants of DLN and spleen cells stimulated with anti-CD3 and anti-CD28 were compared, there was a significant decrease in IL-17A production by DLN cells from rGal-1 treated group as opposed to control mice (Fig. 7I). In contrast, we observed significant increase in IL-10 production by DLN as well as spleen cells from rGal-1 treated group as compared to control treated group (Fig. 7J). Taken together, these data suggest that rGal-1 treatment post P. aeruginosa corneal infection significantly decreases the total number of Th17 cells and IL-17A production at site of infection as well as in lymphoid organs. Interestingly, rGal-1 treatment also increases production of the antiinflammatory cytokine IL-10 by DLN and spleen cells.

FIGURE 7. Local rGal-1 treatment after corneal P. aeruginosa infection modulates Th17 and Treg cells differentially in lymphoid organs.

C57BL/6 mice infected with 3.5 × 10⁵ CFU of P. aeruginosa were therapeutically treated with rGal-1 or control vehicle by local subconjunctival injection from day 1 to 11 pi. Representative FACS plots for IL-17A secreting CD4⁺ T cells on day 12 pi DLN (A) and spleen (B) cells stimulated with anti-CD3/anti-CD28 for 5 hrs from control (left) and rGal-1 treated (right) group are shown. Representative FACS plots for Foxp3⁺CD4⁺ T cells on day 12 pi DLN (C) and spleen (D) from control (left) and rGal-1 treated (right) animal group are shown. Total cell numbers for CD4⁺ T cells, IL-17A⁺ CD4⁺ T cells and Foxp3⁺ T cells on day 12 pi in DLN (E) and spleen (F) from control (clear bar) and rGal-1 treated (grey bar) groups. Cell ratios for total numbers of Treg per Th17 cell in DLN (G) and spleen (H) from control (clear bar) and rGal-1 treated (grey bar) groups. IL-17A (I) and IL-10 (J) protein levels analyzed by sandwich ELISA from P. aeruginosa-infected rGal-1 treated and control DLN and spleen cells harvested at day 12 pi and stimulated with anti-CD3 / anti-CD28 for 48 hrs. Data are a representative of three independent experiments and show mean values ± SEM (n = 12–14). Statistical levels of significance were analyzed by the Student t test (unpaired).

rGal-1 treatment shifts the balance from proinflammatory Th17 to antiinflammatory Th2 response in secondary lymphoid organs

Past studies have indicated that C57BL/6 mice are more susceptible to P. aeruginosa-induced corneal keratitis as compared to BALB/C mice (13). This increased susceptibility of B6 mice was attributed to a dominant Th1 cell-mediated immune response, whereas the resistant phenotype in BALB/c mice was due to a strong Th2 cells-mediated immune response (13, 14, 16). Furthermore, shifting the balance from Th1 to Th2 immune response in B6 mice diminishes the susceptibility to P. aeruginosa-induced corneal pathology indicating that a Th2 biased response protects mice from severe keratitis (15, 16). Interestingly, Gal-1 has been shown to mediate its antiinflammatory effect by suppressing Th1 and Th17, while promoting Th2 response (35, 42, 50). To assess whether rGal-1 treatment during P. aeruginosa-induced bacterial keratitis affects the severity of corneal pathology by shifting the proinflammatory Th17 response towards an antiinflammatory Th2 response, we evaluated the relative composition of Th2 cells in DLN and spleen of rGal-1 treated mice as compared to control treated mice on day 12 pi. The results shown in Fig. 8A–B clearly indicate that the rGal-1 treatment resulted in increased percentage of Th2 cell population in both DLN and spleen as compared to control treated mice. Moreover, the ratio of total number of Th2 cells per Th17 cell was significantly increased in DLN and spleen of rGal-1treated mice as compared to control mice (Fig. 8C–D). Also, the cell supernatants of DLN and spleen cells stimulated with anti-CD3 and anti-CD28 showed a significant increase in IL-4 production by DLN cells from rGal-1 treated group as opposed to control mice (Fig. 8E). However, there was no significant change in IL-4 production by spleen cells (Fig. 8F). Collectively, these results suggest that rGal-1 treatment after corneal P. aeruginosa infection modulates corneal immunopathology by suppressing proinflammatory Th17 immune response and promoting protective Th2 cell response in secondary lymphoid organs.

FIGURE 8. rGal-1 treatment after corneal P. aeruginosa promotes Th2 response in lymphoid organs.

C57BL/6 mice infected with 3.5 × 10⁵ CFU of P. aeruginosa were treated with rGal-1 or control vehicle by local subconjunctival injections from day 1 to 11 pi. Representative FACS plots for IL-4 secreting CD4⁺ T cells on day 12 pi DLN (A) and spleen (B) cells stimulated with anti-CD3/anti-CD28 for 5 hrs from control (left) and rGal-1 treated (right) animal group are shown. Cell ratios for total numbers of Th2 cells per Th17 cell in DLN (C) and spleen (D) on day 12 pi from control (left) and rGal-1 treated (right) group. Data are representative summary of two independent experiments and show mean values ± SEM (n = 8). IL-4 protein levels analyzed by ELISA from P. aeruginosa infected rGal-1 treated and control DLN (E) and spleen (F) cells harvested at day 12 pi and stimulated with anti-CD3 / anti-CD28 for 48 hrs. Data are representative summary of three independent experiments and show mean values ± SEM (n = 12). Statistical levels of significance were analyzed by the Student t test (unpaired).

Discussion

In this report, we have shown for the first time that Th17 cells predominate over Th1 cells and can orchestrate various critical events involved in the pathogenesis of corneal P. aeruginosa infection. Our data also indicate that Treg cells migrate into the cornea after infection during acute and chronic stages of corneal pathology. Interestingly, treatment of P. aeruginosa infected mice with Gal-1, an endogenously derived immunoregulatory molecule, significantly diminishes the number of pathogenic Th17 cells and neutrophils with a concomitant reduction in corneal pathology. Furthermore, Gal-1 shifts the balance from proinflammatory Th17 cells towards Th2 cells indicating the potential therapeutic value of Gal-1 in vision impairing P. aeruginosa-induced bacterial keratitis.

Corneal transparency is an essential prerequisite for optimal vision. However, corneal infection with various pathogens such as P. aeruginosa induces a strong immunoinflammatory reaction characterized by severe alteration in corneal tissue architecture and vision loss (4). Past studies have shown the critical role of CD4⁺ T cells, particularly Th1, in the pathogenesis of P. aeruginosa-induced bacterial keratitis (12–14). However, the actual corneal damage is mediated by the persistent migration of neutrophils into corneal stroma and their release of various proinflammatory molecules such as metalloproteinases and free oxy-radicals (4, 11, 17, 18). The migration of neutrophils into corneal stroma after P. aeruginosa infection is mainly mediated through chemokines, MIP-2 and CXCL1 (4, 18). However, numerous factors such as cytokines and growth factors control the essential steps for neutrophil migration, activation and function at the site of inflammation (20, 52). Accordingly, the recent discovery of IL-17A and its central role in migration and activation of neutrophils indicate a possible involvement of IL-17A in corneal P. aeruginosa infection (19–22). Indeed, a recent study showed that IL-17A expression was increased after corneal P. aeruginosa infection and neutralization of IL-17A significantly diminished bacterial burden and corneal pathology (23). Moreover, mice lacking the receptor for IL-17A (IL-17 receptor A knock-out or IL-17RAKO) were resistant to corneal pathology as compared to wild type control mice (23). Although this study indicated the critical role of IL-17A, the source of IL-17A as well the role of Th17 and Treg populations were not defined. Our data demonstrate that Th17 cells migrate into the cornea as early as day 5 pi with increased numbers as the disease progresses to a more severe form. These findings, in conjunction with published studies showing that IL-17A plays a central role in the migration and activation of neutrophils (19–22), lead us to propose that an increase in the number of Th17 cells in P. aeruginosa infected corneas most likely contributes to corneal pathology through increased migration of neutrophils at the site of inflammation.

IL-17A is a proinflammatory cytokine secreted by various cells such as Th17 cells, γδ T cells, NK cells, NKT cells, neutrophils and mast cells (19–22). IL-17A activates epithelial, endothelial and stromal cells to secrete various chemokines (CXCL1, MIP2), cytokines (IL-6), matrix metalloproteinases (MMP-1, MMP-3, MMP-9, MMP-13), and growth factors (G-CSF, GM-CSF, VEGF-A) essential for migration and activation of various immune cell types at the site of inflammation (20, 53–58). Although our data indicate the involvement of Th17 cells in P. aeruginosa-induced corneal pathology, further studies are required to determine the source of IL-17A early after corneal infection. In addition to Th17 cells, other cell types such as γδ T cells, macrophage and neutrophils can act as a potential source of IL-17A after corneal P. aeruginosa infection. In this respect, it has been shown that innate γδ T cells infiltrate the cornea and produce IL-17A after corneal HSV infection (46). In pulmonary infection by P. aeruginosa, γδ T cells have been shown to promote IL-17A production during the innate immune response (59). Thus, it is possible that one or multiple cell types could contribute to the early source of IL-17A after corneal P. aeruginosa infection. Another important issue that needs to be addressed is why corneal P. aeruginosa infection induces a strong Th17 cell response compared to Th1 cells. Th17 cells differentiate from naïve CD4⁺ T cells after cognate antigen stimulation in the presence of IL-6, TGF-β, IL-21 and IL-23 and are characterized by a lineage specific transcription factor, RORγt (19–22). Previous studies have shown that corneal epithelial cells express various Toll-like receptors (TLR), and ligation of TLR5 with flagellin on corneal epithelial cells induces expression of IL-6, IL-23, IL-1β and TGF-β (60–62). Thus corneal P. aeruginosa infection through flagellin-mediated activation of TLR5 on corneal epithelial cells may generate a local cytokine milieu conducive for Th17 induction and differentiation. Although we have not determined antigen specificity of the Th17 cells, it is possible that these cells recognize one or more antigens from the bacterial outer cell membrane or secreted proteins. In support, one study using live attenuated P. aeruginosa vaccine identified three bacterial proteins OprL, PopB and FpvA that induced a strong Th17 response (63). However, further studies are needed to confirm the antigen specificity of Th17 cells in corneal immunopathology.

Past studies using the mouse model of lung P. aeruginosa infection have shown that the Th17 response is induced after bacterial infection and this contributes to either protection or pathology depending on the severity of infection. Accordingly, acute pulmonary P. aeruginosa infection induces IL-17A response and Th17 cells act as a source of IL-17A (64). This study showed that IL-17A protects mice from acute pulmonary infection through increased migration of neutrophils and subsequent control of bacterial load (64). Furthermore, IL-17A has been shown to play an important role in vaccine-induced protection against acute lung infection by LPS-heterologous strains of P. aeruginosa (65). On the other hand, cystic fibrosis, a chronic lung infection with P. aeruginosa, induces a strong IL-17A and IL-23 response and plays an important role in airway damage and lung inflammation through increased migration of neutrophils (66, 67). However, data from our study as well as others indicate that IL-17A and the Th17 response in the cornea appears to be pathogenic and may contribute to the corneal tissue damage and vision loss (23, 46, 57, 68, 69). Accordingly, HSV infection of the cornea induces a biphasic IL-17A expression and contributes to the corneal pathology and corneal neovascularization through increased migration of neutrophils, increased expression of proinflammatory cytokines as well as proangiogenic molecules (46, 57, 70). Moreover, dry eye disease induces strong IL-17A/Th17 response through increased production of IL-1β, IL-6 and TNF-α by corneal epithelial cells and contributes to the corneal pathology by increasing epithelial cell expression of MMP-3 and MMP-9 and subsequent increased corneal permeability (71). Collectively, data from these studies indicate that the corneal surface, particularly epithelial cells, is endowed with capabilities such as expression of TLRs as well as production of various proinflammatory cytokines essential for generation of strong Th17 biased immune response after inflammatory stimulus.

The recent discovery of suppressor Treg cells and their critical role in suppressing Th1 and Th17 effector T cells represent a promising therapeutic target in various autoimmune and inflammatory disorders (24, 25, 30, 47). Accordingly, shifting the balance from an effector T cell response towards a regulatory T cell response has shown promising therapeutic outcomes in murine models of various autoimmune and chronic inflammatory disorders (24, 25, 27, 30). In addition, different host derived endogenous molecules have been identified that control the excessive effector T cell response and promote Treg response (28, 30). One such family of carbohydrate-binding proteins, galectins, have been identified and characterized, which play an important role in the regulation of both innate and adaptive immune response (28, 31–33). Gal-1 was the first identified member of this family. It plays an important immunomodulatory role in various autoimmune and pathological disorders (28, 33). Gal-1 is prominently expressed at the site of T cell activation as well as immune privileged sites and is secreted by a range of different innate and effector cells (28, 33). Furthermore, Gal-1 controls excessive immune response and host tissue damage by suppressing effector T cell differentiation and proliferation, promoting increased apoptosis of effector T cells, increased generation of Treg cells and increased synthesis of antiinflammatory molecules (28, 33). Additionally, Gal-1 induces tolerogenic DCs through increased synthesis of IL-27, which, in turn, promotes induction of Tr1 cells (28, 33, 72). Moreover, it has been shown that Gal-1 can promote IL-10 synthesis by CD4⁺ T cells during differentiation or after lineage commitment to Th1 and Th17 cells through induction of transcription factors c-Maf and aryl hydrocarbon receptor (39). Recently, Toscano et al. (42) showed the therapeutic potential of Gal-1 in experimental autoimmune uveitis where administration of rGal-1 significantly diminished retinal pathology by shifting the balance from uveitogenic Th1 response toward a nonpathogenic antiinflammatory Th2 and Treg response. Similarly, rGal-1 treatment has been shown to diminish HSV-induced Th1-mediated corneal immunopathology by shifting the balance between proinflammatory cytokine and effector T cells towards protective antiinflammatory cytokines and Treg cells (36). Indeed, our data demonstrate that rGal-1 treatment significantly diminishes corneal pathology via a combined effect of reduced infiltration of neutrophils, reduced production of proinflammatory cytokines as well as a reduced number of Th17 cells in both cornea and local DLN. Furthermore, our data also suggest that rGal-1 treatment shifts the balance from Th17 response towards Th2 and regulatory T cell response.

In conclusion, our data demonstrate that Th17 cells are dominant over Th1 cells during corneal P. aeruginosa infection. Although a Treg response is also generated after P. aeruginosa infection, the ratio of Treg to Th17 diminishes as infection progresses to a severe corneal pathology. Our results also indicate for the first time that corneal pathology post P. aeruginosa infection can be controlled by administration of host-derived endogenous molecules such as Gal-1. Currently, therapies are mainly targeted to control the bacterial burden. However, damage to the cornea and associated vision loss is primarily due to host immune system’s overt reaction to P. aeruginosa infection. Therefore, therapies targeting inflammatory reaction to minimize immunopathology associated corneal damage may prove to be a better therapeutic approach to control vision loss associated with P. aeruginosa keratitis, an important cause of human blindness worldwide.

Abbreviations used in this article

Gal-1

galectin-1

rGal-1

recombinant galectin-1

P. aeruginosa

Pseudomonas aeruginosa

DLN

draining lymph node

Foxp3

forkhead box P3

pi.

postinfection

Treg

regulatory T cells