IL-17 Promotes Pseudomonas aeruginosa Keratitis in C57BL/6 Mouse Corneas

Rao Me, Nan Gao, Chenyang Dai and Fu-shin X. Yu
J Immunol January 1, 2020, 204 (1) 169-179; DOI: https://doi.org/10.4049/jimmunol.1900736

Key Points

  • IL-23/17/17R signaling is upregulated in P. aeruginosa keratitis.

  • IL-17 suppresses TH2 response, upregulates osteoprotegerin, and worsens keratitis.

  • IL-17 could be a therapeutic target for treating P. aeruginosa keratitis.

Abstract

The aim of this study was to elucidate the expression and functions of IL-17 in C57BL/6 mouse corneas in response to Pseudomonas aeruginosa infection. We found that P. aeruginosa infection induced and increased signaling of IL-23/23R/17/17R in mouse corneas. Targeting IL-17A or the IL-17A–specific receptor IL-17RA/IL-17RC with neutralizing Abs resulted in a significant decrease in the severity of P. aeruginosa keratitis, including a decrease in bacterial burden and polymorphonuclear leukocyte infiltration. IL-17A–signaling blockade also significantly reduced the expression of the proinflammatory cytokines L-1β, IL-24, and MMP-13 and increased the expression of the anti-inflammatory cytokine IL-1RA in mouse corneal epithelium. The presence of mouse IL-17A exacerbated P. aeruginosa–mediated tissue destruction. A cytokine protein array revealed that the expression of osteoprotegerin (OPG) was regulated by IL-17A, and OPG neutralization also resulted in a decrease in the severity of P. aeruginosa keratitis. Although both IL-17 and OPG affected the balanced expression of IL-1β and IL-1RA, only IL-17 inhibited the expression of TH2 cytokines. Taken together, our results revealed that IL-17A, along with its downstream factor OPG, plays a detrimental role in the pathogenesis of P. aeruginosa keratitis. Targeting IL-17A and/or the OPG/RANKL/RANK/TRAIL system is a potential therapeutic strategy in controlling the outcome of P. aeruginosa keratitis, which was demonstrated by concurrent topical application of IL-17A–neutralizing Ab and ciprofloxacin in B6 mice.

Introduction

Microbial keratitis is a sight-threatening disease that occurs worldwide. It remains one of the major causes of irreversible corneal blindness, which is the second most common global cause of legal blindness after cataracts (1). Contact lens use is a significant predisposing risk factor for microbial keratitis, especially in patients with extended-use lenses (2). Corneal hypoxia, decreased tear production, microtrauma, and increased cornea temperature caused by the contact lens allows pathogens to better adhere to the ocular surface and increases their opportunity to infect the eye (3, 4). Among all contact lens-related pathogens, Pseudomonas aeruginosa is the most frequently isolated and most pathogenic organism (5). P. aeruginosa causes a keratitis with rapid onset and progression and clinically manifests with strong inflammation and ulceration. More severe complications, including anterior chamber hypopyon and descemetocele formation, corneal scarring, and perforation, may occur (6).

The severe keratitis caused by P. aeruginosa is known to result from not only the high-virulence characteristics of the bacteria itself, but also from the excessive host immune inflammatory response (7). P. aeruginosa can quickly attach to the corneal epithelium by its pili and then inject various toxins to the host cell using the type III system (8). Elastase and alkaline proteases produced by P. aeruginosa can also disrupt the epithelium barrier and promote invasion to the corneal stroma (8, 9). Our previous study showed that, in a B6 mouse model of P. aeruginosa keratitis, it takes ∼18–24 h for the bacteria to cross the epithelial basement membrane and reach the stroma (10). Certain components of the bacteria can activate the innate defense system. For example, the corneas, as well as the lung pretreated with P. aeruginosa flagellin, significantly attenuated the severity of infectious diseases by activating TLR5 signaling and reprogramming the expression of downstream genes to enhance the innate defense function (11, 12). However, the immune response to P. aeruginosa invasion is not always protective, and an overwhelming host inflammatory response can cause tissue destruction (13, 14). For example, neutrophils are crucial for bacterial clearance, but persistent neutrophil recruitment and degranulation releases excessive oxidants, including hydrogen peroxide and hydroxyl radicals, that attack host tissues (15). Inflamed leukocytes also secrete proteolytic enzymes that damage host structures (16). Several studies showed that depletion of specific proinflammatory mediators, such as IL-1β, promote bacterial clearance in P. aeruginosa keratitis (17, 18). The need for a balanced host response to infection is also important in other mucosal tissues, such as the lung and in the cornea (19, 20). In general, rapid resolution of infection-induced inflammation in a tissue, such as the cornea, is determined by the balance of pro- and anti-inflammatory immune responses, which include the expression of cytokines and chemokines, of which one such group is the IL-17 family of cytokines (14).

IL-17 was first identified in 1993 and rose to prominence after the discovery of IL-17–secreting CD4+ T cells in 2005 (21). There are six members in the IL-17 cytokine family, IL-17A–F (22). IL-17A is the prototypical member of this family, and signals through a heteromeric receptor complex consisting of IL-17RA and IL-17RC, along with its homolog IL-17F. Other family members signal through multimeric units, sharing the common chain IL-17RA (23, 24). Although Th17 cells are a major source of IL-17 cytokines, they can also be produced by innate immune cells, including dendritic cells (DCs), macrophages, γδ T cells, and type 3 innate lymphoid cells (25). Binding of IL-17AR recruits the adaptor Act1, which is also an E3 ubiquitin ligase, and activates TRAF-6, which initiates the NF-κb and MAPK pathways and starts the transcription of many downstream genes (26–28). IL-17 has been shown to play a role in both pathological states and homeostasis of mucosal tissues. Proper IL-17 signaling enhances the immunity that protects the host from pathogen invasion (29). For example, IL-17 has been shown to play a crucial role in protection against fungal infection (30, 31). Patients who have IL-17 genetic defect are much more prone to have mucocutaneous candidiasis (32). Moreover, IL-17–deficient mice are shown to be more susceptible to Klebsiella pneumoniae and Streptococcus pneumoniae (33, 34). In contrast, unrestrained IL-17 signaling can lead to immunopathology and inflammation-induced tissue destruction (35, 36). IL-17 also has also been linked to the pathogenesis of many autoimmune diseases, including psoriasis, inflammatory bowel disease, and autoimmune arthritis (37–39). In short, IL-17A acts as a double-edged sword, promoting an immune response that can defend against infection but may also result in damage to the host.

Given the dual roles of IL-17A in mucosal immunity, we assessed the expression of IL-17 signaling and its role in P. aeruginosa keratitis of B6 mice. We found that the IL-23/17 signaling was greatly elevated in the P. aeruginosa–infected mouse cornea. Blockade of IL-17/17R signal significantly attenuated the severity of P. aeruginosa keratitis by altering gene expression and suppressing infection-induced inflammation. We also identified that osteoprotegerin (OPG) is a downstream effector of IL-17 signaling and plays a detrimental role in P. aeruginosa corneal infections. Targeting IL-17 and/or OPG may be used as an adjunctive therapy, in combination with antibiotics, to treat bacterial keratitis. Concurrent topical application of IL-17A–neutralizing Ab and ciprofloxacin demonstrated therapeutic potential of IL-17 neutralization as an adjunctive therapy for treating P. aeruginosa keratitis in B6 mice.

Materials and Methods

Animals

Wild-type C57BL/6 (B6) mice (8 wk old; female) were purchased from The Jackson Laboratory (Bar Harbor, ME). All animal procedures were performed in compliance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Visual Research and were approved by the Institutional Animal Care and Use Committee of Wayne State University.

Mouse model of P. aeruginosa keratitis

Mice were anesthetized with an i.p. injection of ketamine (90 mg/kg) and xylazine (10 mg/kg) before surgical procedures. Mouse corneas were scratched gently with a sterile 26-gauge needle to create three 1-mm incisions to break the epithelial barrier and were inoculated with 1.0 × 104 CFU of ATCC 19660, a virulent laboratory strain known to consistently produce severe keratitis in experimentally infected mice with type III secretion system, in 5 μl of PBS.

Administration of neutralizing Ab or recombinant protein

To apply neutralizing Abs or recombinant proteins, mice were subconjunctivally injected with anti–IL-17 (250 ng/5 μl; R&D Systems, Minneapolis, MN), anti-17RA (400 ng/5 μl; R&D Systems), anti-17RC (400 ng/5 μl; R&D Systems), and recombinant mouse (rm)–IL-17A (200 ng/5μl; R&D Systems) 4 h before the inoculation with P. aeruginosa on the corneas. To explore the clinical use of anti–IL-17 treatment, ciprofloxacin ophthalmic solution was used to dissolve anti–IL-17 Ab, and 5 μl was instilled into mouse corneas, starting 16 h after P. aeruginosa inoculation and continuing every 2 h thereafter during days 1 and 2 and every 4 h during day 3 after initial treatment.

Isolation of mouse corneal epithelial cells

A razor blade was tailored to ∼5 mm wide in the edge and placed in a Castroviejo razor blade breaker and holder. Mice were euthanized by cervical dislocation. Under the microscope, corneal epithelial cells (CECs) were surgically scraped off from the basement membrane. Cells were collected to the razor blade from the basement membrane. Liquid nitrogen was used to snap freeze the cells and cool off the tip of a sharp surgical scalper at the same time. Cells were immediately transferred into precooled 1.5-ml Eppendorf tubes placed on dry ice by scraping the razor blade with the scalper. Cells were processed immediately for RNA isolation or protein extraction, or they were stored at −80°C for later use.

Clinical examination, quantification of P. aeruginosa CFU, and determination of myeloperoxidase units

Corneas were photographed at 1 d postinfection (dpi) for the assessment of infection severity. Clinical scores were assigned to the infected corneas in a blinded fashion according to a previously reported scale (40). Whole corneas were excised and placed in 200 ml of sterile PBS. Tissue was homogenized with a TissueLyser II (QIAGEN, Valencia, CA). The homogenates were divided into two parts. The first fraction (50 ml) was subjected to serial log dilutions for the assessment of viable bacterial number. The remaining homogenates were further lysed for myeloperoxidase (MPO) measurement. MPO units were determined according to a previously reported method. One MPO unit corresponds to 2.0 × 105 polymorphonuclear leukocytes (PMNs).

Semiquantitative and quantitative PCR

The primers used in this study are listed in Table I. Total RNA was extracted with an RNeasy Mini Kit (QIAGEN) following the manufacturer’s instructions. For semiquantitative PCR, cDNA was amplified with TaqMan technology (Promega, Madison, WI). PCR products were subjected to electrophoresis on 2% agarose gels containing ethidium bromide. For quantitative PCR (q-PCR), cDNA was amplified using a StepOnePlus Real-Time PCR System (Applied Biosystems, University Park, IL) with SYBR Green PCR Master Mix (Applied Biosystems). Data were analyzed by using the ΔΔ cycle threshold method with β-actin as the internal control.

Western blot, ELISA, and protein array

Mouse corneal samples were lysed with radioimmunoprecipitation assay buffer. The lysates were centrifuged to obtain supernatant. Protein concentration was determined by bicinchoninic acid assay. For Western blot analysis, the protein samples were separated by SDS-PAGE and electrically transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 5% milk and subsequently incubated with primary and secondary Abs. Signals were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Pittsburgh, P. aeruginosa). β-Actin was used as the loading control. Quantification of protein levels was based on the densitometry of blots by using the software Carestream Molecular Imaging Software (Informer Technologies, Rochester, NY). The Abs used included the following: anti–IL-23, anti–IL-23R, anti–IL-17A, anti–IL-17RC, anti–MCPIP-1, anti-OPG (R&D Systems), and anti–β-actin (A1978; Sigma-Aldrich). ELISA (IL-23; R&D Systems) and protein array (Proteome Profiler Array Mouse XL Cytokine Array Kit; R&D Systems) were performed following manufacturer’s protocols.

Immunohistochemistry

At the indicated time points, the corneas were enucleated, embedded in Tissue-Tek O.C.T. Compound, and frozen in liquid nitrogen. Six-micrometer-thick sections were cut and mounted to poly-l-lysine–coated glass slides, fixed in 4% paraformaldehyde, and blocked with PBS containing 2% BSA for 1 h at room temperature. Sections were then incubated with primary Abs rat anti-mouse NIMP-R14 (1:50; BD Biosciences), followed by the secondary Ab, FITC-conjugated goat anti-rabbit IgG (1:100; Jackson ImmunoResearch Laboratories). Slides were mounted with VECTASHIELD Mounting Media containing DAPI. Controls were similarly treated with corresponding IgG from the same animal as the primary Ab.

Flow cytometry analysis

Whole corneas were digested in 20 μl Liberase TL (2.5 mg/ml; Sigma-Aldrich), followed by incubation at 37°C for 45 min. Cell suspensions were passed through a 70-μm filter. Viable cells were then counted using trypan blue dye exclusion. Cells were incubated at 4°C in PBS containing 2% FBS and Fc. The cells were subsequently labeled with PerCP–Cy5-conjugated CD45, FITC-conjugated Ly-6G, APC-conjugated IL-17RA, or APC-conjugated IL-17RC (eBioscience) for 30 min in 2% FBS at 4°C in the dark. All samples were washed and reconstituted in PBS. Flow cytometry was performed with an FACS system (BD FACSAria II), and the data were analyzed using FlowJo software.

Statistical analysis

A nonparametric Mann–Whitney U test was used to compare the clinical scores. A paired t test or one-way ANOVA was used to compare quantitative means. A p value <0.05 was considered to be significant.

Results

P. aeruginosa infection increases IL-23/17 axis signaling in B6 mouse cornea

To understand the role of the IL-23/17 signaling axis, we first investigated the expression of the IL-23/17 signaling axis in B6 mouse corneas in response to P. aeruginosa infection (Fig. 1). At the mRNA levels, IL-23 and its receptor IL-23R, IL-17A and its receptor IL-17RA, and IL-17RC were significantly increased in mouse corneas at 1 dpi relative to naive corneas, as shown by RT-PCR (Fig. 1A). At the protein level, Western blot analysis showed that, at 1 dpi, there was an increased expression of IL-23, IL-17A, and their receptors IL-23R and IL-17RC (Fig. 1B) in the infected corneas. The time course study of IL-23 expression was performed and shown in Supplemental Fig. 1, and the expression of IL-17RA and -RC in isolated CECs of naive corneas in Supplemental Fig. 2.

FIGURE 1.
FIGURE 1.

P. aeruginosa infection increases IL-23/17 axis signaling in B6 mouse cornea. (A) Mouse corneas were scratched with a needle and inoculated with 1.0 × 104 CFU P. aeruginosa. Whole corneas were collected at 1 dpi for quantitative real-time PCR or semiquantitative RT-PCR analysis of IL-23, IL-17A, IL-17RA, and IL-17RC (n = 3). (B) Western-blot analysis of IL-23, IL-23R, IL-17A, and IL-17RC in cell lysates of whole corneas infected (Inf) with P. aeruginosa at 1 dpi. β-Actin serves as the loading control. The results are presented as a relative increase (fold) to those of naive corneas, set as 1. Data are representative of three independent experiments (A). Mean ± SEM). *p < 0.05 (one-way ANOVA). (C) The corneas were excised and processed for immunohistochemistry analysis at 1 dpi. The 6-μm cryostat sections were stained with anti-IL-17RA (green), anti-IL-23R (green), and DAPI (blue) for nuclei. Two independent experiments were performed; one representative image for each condition is presented. Original magnification ×20. E, epithelium; S, stroma. (D) Flow cytometric analyses of IL-17RA– and IL-17RC–positive immune cells in naive and Inf corneas. Ten corneas were pooled for each sample. Percentage of IL-17RA–, IL-17RC–, and Ly-6G–positive cells are shown in the flow cytometric plots.

Tissue distribution of IL-23 and 17R were assessed using immunohistochemistry. In P. aeruginosa–infected corneas, numerous IL-23R–positive and IL-17RA–positive cells were seen in the corneal stroma (Fig. 1C). Western blot shows mouse corneal epithelium also express IL-17RA and IL-17RC. As most cells in the stroma of 1 dpi corneas were IL-17R positive, we used flow cytometry to determine if neutrophils, the most popular infiltrated cells at early stage of infection, express IL-17RA and IL-17RC (Fig. 1D). Our data showed that in P. aeruginosa–infected corneas, 70.6% Ly-6G–positive cells were IL-17RA positive and 75.6% were IL-17RC positive, consistent with that reported for Aspergillus-induced keratitis (41). Taken together, components of the IL-23/17–signaling pathway were upregulated in P. aeruginosa–infected mouse corneas.

Blockade of IL-17AR improves the outcome of P. aeruginosa keratitis in mouse cornea

Having identified the increased expression of IL-17A receptors IL-17RA and IL-17RC during P. aeruginosa infection in mouse corneas, we next investigated the effect of IL-17AR signaling in the pathogenesis of P. aeruginosa keratitis (Fig. 2). IL-17RA and IL-17RC are heteromeric receptor complex components for IL-17A. Although IL-17RC determines the specificity of IL-17A, IL-17RA is the shared receptor for IL-17 cytokines. As shown in Fig. 2, treating the corneas with IL-17RA– or IL-17RC–neutralizing Abs resulted in a significant decrease in the severity of P. aeruginosa keratitis, including markedly reduced clinical scores, significantly dampened bacterial burden, and notably decreased MPO activities. Hence, both IL-17RA and IL-17RC participate in the pathogenesis of P. aeruginosa keratitis in B6 mouse corneas.

FIGURE 2.
FIGURE 2.

IL-17RA– and IL-17RC–neutralizing Abs decrease the severity of P. aeruginosa infection in B6 mouse cornea. Mice were subconjunctivally injected with IL-17RA (400 ng/5 μl)– and IL-17RC (400 ng/5 μl)–neutralizing Ab 4 h before the inoculation with 1.0 × 104 CFU P. aeruginosa. Mouse IgG serves as control. (A) Mouse corneas were monitored and photographed (original magnification ×10) at 1 dpi. The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. (B and C) At 1 dpi, the corneas were excised and subjected to bacterial plate counting (CFU per cornea) and MPO unit determination. Data are representative of three independent experiments with five corneas per group (mean ± SEM) (n = 5). **p < 0.01 (paired t test).

IL-17A promotes P. aeruginosa keratitis in B6 mouse cornea

Having shown no detectable differences between blocking IL-17ARA and -RC at 1 dpi, we next investigated the effects of IL-17A activity in P. aeruginosa keratitis using two complementary approaches: application of IL-17A–neutralizing Ab or application of exogenous mouse IL-17A prior to P. aeruginosa inoculation. Neutralizing Ab was subconjunctivally injected 4 h before P. aeruginosa inoculation. Blockade of IL-17A resulted in much-reduced severity of P. aeruginosa keratitis, compared with those eyes injected with control IgG (Fig. 3). The clinical scores assigned to anti–IL-17A mice were significantly lower than those of control IgG group with lowered bacterial burden and MPO activity (Fig. 3B, 3C).

FIGURE 3.
FIGURE 3.

IL-17A–neutralizing Ab and rm–IL-17 have opposing effects on the outcome of P. aeruginosa infection in B6 mouse corneas. Mice were subconjunctivally injected with IL-17A–neutralizing Ab (250 ng/5 μl) or rm–IL-17A (200 ng/5 μl in 0.1% BSA) 4 h before the inoculation with 1.0 × 104 CFU P. aeruginosa. Mouse IgG or 0.1% BSA serves as control. (A and D) Mouse corneas were monitored and photographed (original magnification ×10) at 1 dpi. The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. (B, C, E, and F) At 1 dpi, the corneas were excised and subjected to bacterial plate counting (CFU per cornea) and MPO unit determination. Data are representative of three independent experiments with five corneas per group (B and C) (mean ± SEM; n = 5). *p < 0.05, **p < 0.01 (paired t test). (G) Mouse corneas were treated with anti-IL-17 Ab or rm–IL-17A and inoculated with P. aeruginosa. Naive corneas were used as negative control. The corneas were excised and processed for immunohistochemistry analysis at 1 dpi. The 6-μm cryostat sections were stained with NIMP-R14 Ab for neutrophils. The images of neutrophils (green) were merged with DAPI (blue nuclei) staining. Two independent experiments were performed; one representative image for each condition is presented. Original magnification ×10. E, epithelium; NL, naive cornea; S, stroma.

We next administrated rm–IL-17A prior to P. aeruginosa inoculation. In contrast to blockade of IL-17A, the presence of exogenous IL-17A markedly increased the susceptibility of mouse corneas to P. aeruginosa infection, with higher clinical scores (Fig. 3D), bacterial burden, and MPO activity compared with the BSA control group (Fig. 3D, 3F).

Immunofluorescence analysis revealed the role of IL-17A in the neutrophil recruitment in P. aeruginosa–infected corneas (Fig. 3G). No neutrophil staining can be detected in the naive corneas. Numerous Ly-6G–positive cells were observed in corneal stroma in P. aeruginosa–infected corneas, consistent with the increased MPO activity. In contrast to IL-17A blockade, which decreased the number of infiltrated neutrophils, exogenous rm–IL-17A greatly increased the number of infiltrated neutrophils in the cornea, with epithelium edema and heavy stromal infiltration. Hence, blockade of IL-17A decreased and presence of rm–IL-17A increased the severity of P. aeruginosa keratitis in B6 mouse corneas.

Blockade of IL-17A altered gene expression in response to P. aeruginosa infection in B6 mouse corneas

We next used IL-17A–neutralizing Abs and real-time PCR to assess the effects of IL-17A on the expression of several innate immune responsive genes, which were shown to be associated with the pathogenesis of P. aeruginosa keratitis (Fig. 4, Table I). At 6 h postinfection (hpi), IL-17A blockade dampened expression of CXCL-1, IL-24, and MMP13 and increased expression of the anti-inflammatory gene IL-10 and IL-1Ra in response to P. aeruginosa infection in B6 mouse corneas (Fig. 4). Importantly, both S100A8 and A9 were highly induced in CECs in response to P. aeruginosa in an IL-17A–independent manner.

FIGURE 4.
FIGURE 4.

Blockade of IL-17A alters gene expression in B6 mouse corneas in response to P. aeruginosa infection. Mouse corneas were treated with anti-IL-17A Ab or control IgG and inoculated with 1.0 × 104 CFU P. aeruginosa. CECs were collected at 6 hpi and analyzed by real-time PCR. The results are presented as a relative increase (fold) to those of naive corneas, set as 1. Data are representative of three independent experiments with three corneas per group (mean ± SEM; n = 3). *p < 0.05, **p < 0.01 (one-way ANOVA).

View this table:
Table I. Primer sequences used for PCR

IL-17A promotes P. aeruginosa keratitis in mouse cornea in part by regulating OPG expression

To further explore the underlying mechanism of IL-17 signaling in P. aeruginosa keratitis, we assessed the effects of IL-17A on the expression of cytokines, chemokines, and growth factors using the XL mouse cytokine array. Among 111 genes, OPG protein levels were undetectable in naive corneas and became abundant in P. aeruginosa–infected corneas (Supplemental Fig. 3). IL-17A neutralization suppressed, whereas rm–IL-17A augmented, the infection-induced expression of OPG at 1 dpi (Fig. 5A). To confirm the cytokine array results, quantitative RT-PCR (qRT-PCR) and Western blot were performed. qRT-PCR revealed that OPG transcripts were significantly increased in the P. aeruginosa–infected cornea relative to naive corneas; a significant suppression of OPG transcription was observed in anti–IL-17A–treated corneas, whereas augmented OPG expression was observed in rm–IL-17A–treated corneas (Fig. 5B). Western blot analysis showed a similar pattern of OPG expression at the protein levels in P. aeruginosa–infected corneas with different IL-17A activities (Fig. 5C). Tissue distribution of OPG was also assessed. No staining was detected in naive corneas, whereas strong staining of OPG was observed in the stroma in P. aeruginosa–infected corneas, with minimal staining in the epithelium (Fig. 5D).

FIGURE 5.
FIGURE 5.

IL-17A regulated OPG expression in B6 mouse corneas in response to P. aeruginosa infection. Mouse corneas were treated with anti–IL-17A Ab or rm–IL-17A and inoculated with 1.0 × 104 CFU P. aeruginosa. Whole corneas were collected at 1 dpi. (A) Protein array analysis revealed the effect of IL-17A on cytokine expression. Selected images for OPG was shown. (B) q-PCR analysis of OPG in whole corneas infected with P. aeruginosa at 1 dpi (n = 3). (C) Western blot analysis of OPG in cell lysate of whole corneas infected with P. aeruginosa at 1 dpi. β-Actin serves as the loading control. Data are representative of three independent experiments with three corneas per group (B) (mean ± SEM). **p < 0.01 (one-way ANOVA). (D) The corneas were excised and processed for immunohistochemistry analysis at 1 dpi. The 6-μm cryostat corneal sections were stained with anti-OPG (green) and DAPI (blue) for nuclei. Two independent experiments were performed; one representative image for each condition is presented. Original magnification ×10. E, epithelium; S, stroma.

OPG regulates IL-17A, but not S100A8/9, expression in P. aeruginosa–infected corneas

We next assessed the function of OPG in the pathogenesis of P. aeruginosa keratitis. Blockade of OPG by neutralizing Ab resulted in a decrease in the severity of keratitis, including lower clinical scores, dampened bacterial burden, and reduced influx of neutrophils, as indicated by MPO activity, when compared with those eyes injected with control IgG (Fig. 6). qRT-PCR analysis revealed that OPG neutralization significantly decreased the levels of IL-17A transcripts but exhibited no effects on the expression of the antimicrobial peptides S100A8 and S100A9 in P. aeruginosa–infected corneas (Fig. 6D).

FIGURE 6.
FIGURE 6.

Blockade of OPG attenuates the severity of P. aeruginosa infection in B6 mouse cornea. Mice were subconjunctivally injected with OPG-neutralizing Ab (200 ng/5 μl) 4 h before the inoculation with 1.0 × 104 CFU P. aeruginosa. Mouse IgG serves as control (n = 5). (A) Mouse corneas were monitored and photographed (original magnification ×10) at 1 dpi. The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. (B and C) At 1 dpi, the corneas were excised and subjected to bacterial plate counting (CFU per cornea) and MPO unit determination. (D) Whole corneas were collected at 1 dpi. q-PCR analysis of anti-OPG in whole corneas infected with P. aeruginosa at 1 dpi. Data are representative of three independent experiments with three corneas per group (B–D) (mean ± SEM; n = 3). *p < 0.05, **p < 0.01 (one-way ANOVA).

Upregulation of OPG is partially responsible for IL-17A–exacerbated P. aeruginosa keratitis

To further identify the role of OPG in the IL-17/17R pathway in P. aeruginosa keratitis, we subconjunctivally injected rm–IL-17A– and OPG–neutralizing Abs simultaneously. Neutralization of OPG significantly dampened the severity of P. aeruginosa keratitis, including a partially decreased clinical score, bacterial burden, and MPO activity compared with rm–IL-17A–only-treated P. aeruginosa keratitis infection as the control (Fig. 7). Hence, P. aeruginosa–induced, IL-17–dependent upregulation of OPG is partially responsible for the observation that IL-17A exacerbates P. aeruginosa keratitis in B6 mouse corneas.

FIGURE 7.
FIGURE 7.

OPG upregulation is partially responsible for IL-17–worsened outcome of P. aeruginosa keratitis in B6 mice. Mouse corneas were treated with rm–IL-17A or rm–IL-17A with anti-OPG Ab and inoculated with 1.0 × 104 CFU P. aeruginosa; 0.1% BSA serves as control. (A) Mouse corneas were monitored and photographed (original magnification ×10) at 1 dpi. The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. (B and C) At 1 dpi, the corneas were excised and subjected to bacterial plate counting (CFU per cornea) and MPO unit determination. Data are representative of three independent experiments with five corneas per group (B and C) (mean ± SEM; n = 5). *p < 0.05, **p < 0.01 (one-way ANOVA).

Blockade of either IL-17A or OPG resulted in continuously attenuated inflammation in B6 mouse cornea in response to P. aeruginosa infection

To further investigate the long-term effects of IL-17A and OPG blockade, we subconjunctivally injected either IL-17A– or OPG-neutralizing Abs 4 h prior to inoculation and allowed the infection to continue until 3 dpi, at which time the control corneas had high clinical scores and were near to corneal perforation; hence, experiments were terminated at 3 dpi (Figs. 8, 9). We found that, at this time point, the expression of all cytokines assessed were induced and significantly than that in the naive corneas. The mRNA levels of TH17 (IL-23 and IL-17A) and TH1 (IFN-γ, IL-2, and TNF-α), as well as IL-6 cytokines, were significantly lower in IL-17 and OPG blockaded compared with infected control corneas, with no significant differences between two treated groups; the lower levels of these cytokines may reflect the decreased severity of keratitis. In contrast, TH2 cytokines (IL-4 and IL-5) as well as IL-10 (a regulatory T [Treg] cytokine) were significantly higher in IL-17A, but not OPG-neutralizing, Ab treated, compared with the infection control corneas. The levels of another Treg cytokine, TGF-β, were similar in all three infected corneas. As for inflammation, blockades of IL-17A and OPG greatly decreased IL-1β and significantly increased IL-1Ra expression, consistent with decreased keratitis severity of these treated corneas.

FIGURE 8.
FIGURE 8.

Blockade of either IL-17A or OPG attenuates the severity of P. aeruginosa keratitis in B6 mouse cornea at 3 dpi. Mice were subconjunctivally injected with either IL-17A–neutralizing or OPG-neutralizing Ab 4 h before the inoculation with 1.0 × 104 CFU P. aeruginosa. Mouse IgG serves as control. (A) Mouse corneas were monitored and photographed (original magnification ×10) at 3 dpi. The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. (B and C) At 3 dpi, the corneas were excised and subjected to bacterial plate counting (CFU per cornea) and MPO unit determination (n = 5). *p < 0.05, **p < 0.01 (one-way ANOVA).

FIGURE 9.
FIGURE 9.

Blockade of IL-17A, but not OPG, promotes Th2 response to P. aeruginosa infection in B6 mice. Subconjunctivally injected with either IL-17A–neutralizing or OPG-neutralizing Ab 4 h before the inoculation with 1.0 × 104 CFU P. aeruginosa. Whole corneas were collected at 3 dpi, q-PCR analysis of (A) IL-23/17 signaling cascade and (B) the expressions of Th1, Th2, Th17, Treg, and other cytokines were performed. Data are representative of three independent experiments with three corneas per group (mean ± SEM; n = 3). *p < 0.05, **p < 0.01 (one-way ANOVA).

Adjunct topical IL-17A–neutralizing Ab improved the outcome of keratitis in antibiotic-treated corneas after P. aeruginosa infection

To explore the potential clinical application of anti–IL-17A treatment as an adjunctive therapy, we topically applied ciprofloxacin concurrently with IL-17A–neutralizing Ab, starting at 16 hpi, by which time our previous study showed that the invading pathogens are mostly in the epithelium layer (42). The eyes treated with anti–IL-17A Ab significantly improved the outcome of P. aeruginosa keratitis (Fig. 10). The clinical score in anti–IL-17 group was significantly decreased from day 1 to 3 posttreatment compared with ciprofloxacin control group. Consistent with reduced corneal opacification, additional topical anti–IL-17A also significantly reduced PMN infiltration. Hence, topical anti–IL-17 treatment has potential as an adjunct therapy to Ab for treating P. aeruginosa keratitis.

FIGURE 10.
FIGURE 10.

Concurrent topical application of IL-17A–neutralizing Ab and ciprofloxacin (Cip) eradicates P. aeruginosa infection–associated inflammation in B6 mice. C57BL/6 mouse corneas were inoculated with 1.0 × 104 CFU P. aeruginosa. Topical solution containing Cip was used to dissolve IL-17A–neutralizing Ab. Topical antibiotic with or without anti–IL-17A Ab was then applied, starting 16 hpi, at 2-h intervals during the first and second days of treatment and at 4-h intervals on the third day of treatment. The infected corneas were (A) photographed (original magnification ×4) and (B) scored, and (C) MPO activity assay was performed at the end of experiment. The results are representative of three independent experiments. Data are representative of three independent experiments with five corneas per group (B and C) (mean ± SEM; n = 5). *p < 0.05, **p < 0.01 (paired t test).

Discussion

In this study, we investigated the role of IL-17A and its receptor IL-17RA/IL-17RC in P. aeruginosa keratitis in B6 mouse corneas. We identified that the IL-23/23R–IL-17/17R signaling pathway is upregulated in the cornea in response to P. aeruginosa infection. Functionally, blockade of IL-17A or its receptor IL-17RA or IL-17RC resulted in decreased severity of P. aeruginosa keratitis, decreased bacterial burden, and inhibited the infection-induced inflammation in B6 mouse cornea. Although addition of exogenous IL-17A exacerbated P. aeruginosa keratitis, its blockade dampened the expression of proinflammatory cytokines/chemokines and suppressed PMN infiltration. We also found that IL-17A induced OPG expression in P. aeruginosa–infected corneas, and neutralization of OPG attenuated P. aeruginosa keratitis. The latter also influences IL-17A expression. IL-17, but not OPG, suppresses TH2 immune response, whereas both promote inflammatory response as assessed by IL-1β/IL-1Ra expression. Finally, concurrent topical application of IL-17A–neutralizing Ab and ciprofloxacin eradicated P. aeruginosa infection–associated inflammation and corneal opacification in B6 mice. Taken together, these results suggest that IL-17/17R signaling plays a pathological role in P. aeruginosa keratitis, including the induction of proinflammatory cytokine/chemokine and OPG expression and the suppression of TH2 response.

IL-23 is a heterodimer belonging to the IL-12 family. It is composed of two subunits, IL-23p19 and IL-12p40, and the latter is a common subunit shared with IL-12 (43). IL-23 is an important mediator of tissue inflammation (44). Upon infection, IL-23 is rapidly produced by activated macrophages and/or DCs at the infected site, and DCs are the main source of IL-23 (45). IL-23 produced by residential DCs is likely to be an initial step in the inflammatory cascade that drives the local expression of proinflammatory mediators such as IL-17 and infiltration of innate defense cells such as neutrophils, NK cells, and innate lymphoid cells, most of which are capable of secreting IL-17A as well as IL-22 (25, 46). Our results showed that at 1 dpi, there are an increased expressions of IL-23/17 axis–signaling molecules, including IL-23, IL-23R, IL-17A, and IL-17RA and -RC, and infiltrated neutrophils, which are both IL-17RA and -RC positive in P. aeruginosa–infected corneas, confirming a previous report showing that neutrophils in Aspergillus-induced keratitis express both IL-17RA and -RC (41). These expression and distribution data suggest the involvement of the IL-23/IL-17 axis in corneal innate defense and the pathogenesis of P. aeruginosa keratitis.

An early study showed that topical treatment with polyclonal Abs to IL-17 resulted in significant reductions in corneal pathology and lowered bacterial counts postinfection with six different laboratory or clinical P. aeruginosa strains, including both invasive and cytotoxic strains, and ICAM1 was suggested as a downstream molecule (47). In the airway mucosa, IL-17A promotes inflammation and impairs host defenses in acute and chronic Pseudomonas lung infection (48, 49). In biofilm bacteria–infected patients, the IL-17 levels in the lavage fluids were significantly higher than that in nonbiofilm bacteria–infected patients; the elevated IL-17 was attributed to chronic injuries caused by biofilm infections (50). The effects of IL-17 on P. aeruginosa biofilm formation are unknown. Our study used complimentary approaches, targeting IL-17A and IL-17RA and -RC individually in, and applying rIL-17A to, P. aeruginosa–infected corneas. Our results show that neutralizing IL-17A, IL-17RA, or IL-17RC all significantly reduced, whereas exogenous rIL-17 increased, bacterial burden and inflammation, including elevated expression of inflammatory cytokines and neutrophil influx and/or accumulation, associated with P. aeruginosa infection of corneas, suggesting that IL-17 plays a pathological role in P. aeruginosa keratitis by augmenting the infection-induced inflammation.

How might IL-17A drive corneal inflammatory response to P. aeruginosa infection in the cornea? It is likely to act in the context of the tissue microenvironment and with multiple inflammatory mediators. As such, we assessed the effects of IL-17A on the expression of genes known to be involved in the corneal innate defense against microbial infection. Corneal infection occurs when epithelial barrier function is compromised and opportunistic pathogens such as P. aeruginosa invade the epithelium, which functions as the first line of innate immune defense. To that end, we assessed the epithelium expression of cytokines and found that downregulating IL-17A signaling inhibited expression of pathogenic factors CXCL1, IL-24, and MMP13 and elevated expression of anti-inflammatory cytokine IL-10 in P. aeruginosa–infected corneas at 6 hpi. CXCL1 is a major neutrophil recruitment chemokine and a downstream gene of IL-17 (51), whereas IL-10 is an anti-inflammatory cytokine that counteracts LPS in inducing CXCL1 expression (52). Our previous study showed that IL-24 and its downstream effector SOCS-3 were induced in the corneal epithelium during P. aeruginosa infection, and they were detrimental, as the early expression of SOCS3 may hinder the development of the innate defense apparatus, including inflammation response, resulting in an elevated severity of keratitis (53). We previously showed that MMP-13 is a pathogenic factor that is hijacked by P. aeruginosa to dissolve the protective structures of the cornea, hence, allowing P. aeruginosa to cross the basement membrane, causing stromal keratitis (54). Suppression of IL-17 activity inhibited the expression of MMP-13, restricting invading P. aeruginosa to the epithelium, where many antimicrobial cytokines, including calprotectin (dimer of S100A8/A9), are expressed. Interestingly, IL-17 signaling appears to have no effect on S100A8/A9 expression. Hence, we conclude that IL-17 blockade dampened the expression of proinflammatory cytokines (IL-24, CXCL-1, and MMP-13) and increased the expression of anti-inflammatory genes (IL-10) to promote the resolution of infection-associated inflammation in B6 mouse cornea.

To further explore the mechanisms underlying IL-17A’s influence on P. aeruginosa infection of the cornea, we used a cytokine protein array and observed and confirmed that the levels of OPG at both transcriptional and translational levels were most dramatically elevated in the mouse cornea in response to P. aeruginosa infection, with IL-17 neutralizing suppressing, and exogenous IL-17 promoting, its expression. Functional study revealed that OPG neutralization attenuated the severity of P. aeruginosa keratitis. OPG encoded by Tnfrsf11b is a decoy receptor of RANK, and RANKL/OPG signaling modulates osteoclast function in bone remodeling (55, 56). It was shown that IL-17 disrupts the RANKL/OPG balance in the synovium and promotes bone erosion in murine collagen arthritis (57). In the cornea, OPG is expressed in fibroblasts and was shown to participate in corneal wound healing (54). Macrophages and neutrophils are known to be the source of OPG (58, 59). Our immunofluorescence analysis also revealed its distribution in the stromal infiltrating cells during P. aeruginosa infection. To our knowledge, this is the first report to link OPG expression to corneal infection and IL-17 signaling and to show a detrimental role of the gene in the pathogenesis of P. aeruginosa keratitis. Interestingly, neutralizing OPG partially downregulated IL-17A expression, suggesting a positive feedback loop of IL-17A and OPG expression. Moreover, whereas IL-17A suppresses TH2-type response, OPG exhibited no such effect in P. aeruginosa–infected corneas. Like IL-17A, OPG neutralization exhibited no effects on the expression of S100A8/A9, suggesting OPG may target other aspects of innate defense, such as apoptosis of infected cells. Indeed, in addition to binding RANKL, OPG also binds to and inhibits TRAIL. TRAIL is known to help to defend against microbial infections by inducing apoptosis of infected cells (60). It is plausible that OPG enhances the infection-induced inflammation by increasing IL-17A expression and inhibiting apoptosis of infected cells, resulting in increased pathology in P. aeruginosa keratitis. The link between IL-23 and IL-17 and involvement of OPG–RANK–RANKL and/or OPG–TRAIL pathways in P. aeruginosa keratitis warrant further investigation.

Our data generated using qRT-PCR revealed that IL-17, but not OPG, suppresses TH2 response in P. aeruginosa–infected corneas at 3 dpi, by which the adaptive immunity has begun to develop in the infected corneas. By comparing mice favoring Th1 (C57BL/6) versus Th2 (BALB/c) response development, mice strains favoring development of a Th1-type response are susceptible (cornea perforates), whereas strains favoring Th2 response development are resistant or protective (no corneal perforation) (61). In contrast, in murine models of fungal keratitis, protective immunity was associated with temporal recruitment of IL-17–producing neutrophils, Th17, and Th1 cells and dependent on production of IL-17, but not IFN-γ (62). We showed that neutralizing IL-17 increased TH2 response, resulting in a decrease in the severity of P. aeruginosa keratitis. Furthermore, we showed that targeting IL-17 and OPG augments IL-1RA expression while suppressing the expression of IL-1β, suggesting IL-17 and OPG skew the innate immune apparatus to a proinflammatory status. The effects of IL-17A on TH2-type immune response and on the balance expression of IL-1β and soluble IL-1Ra may be the underlying mechanisms for IL-17/IL-17R to play a detrimental role in P. aeruginosa keratitis.

Finally, we tested therapeutic potential of IL-17 neutralization on tempering down inflammation while the corneas were treated by topically antibiotics. Our previous study showed that treating P. aeruginosa–infected corneas with the fourth generation antibiotics ciprofloxacin within 16–24 h will result in eradication of invading pathogen, whereas inflammation or corneal opacification remain 3 d after Ab treatment (also, see Fig. 10). Our data showed that concurrently treating the infected corneas with ciprofloxacin and IL-17A–neutralizing Ab reduced corneal inflammation associated with P. aeruginosa infection as assessed by clinical scores and MPO determination. We conclude that IL-17 neutralization, such as the use of bimekizumab, which has been shown to be 100% response with 86.7 versus 0% improvement in Psoriasis Area and Severity Index criteria, sustained to week 20 without unexpected safety signals (63) may be safely used as topical adjunctive reagent to treat microbial keratitis.

Taken together, our study demonstrates that an increase in IL-23/17A/17R axis signaling may worsen P. aeruginosa keratitis in B6 mouse corneas. IL-17A functions in the pathogenesis of P. aeruginosa keratitis in part via induction of OPG. Additionally, IL-17A and/or OPG could be a potential therapeutic target for treating P. aeruginosa keratitis.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Institutes of Health/National Eye Institute (NEI) Grants R01EY10869 and EY17960 (to F.-s.X.Y.), NEI Core Grant p30 EY04078 (to Wayne State University), and Research to Prevent Blindness funds (to Kresge Eye Institute).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    CEC
    corneal epithelial cell
    DC
    dendritic cell
    dpi
    day postinfection
    hpi
    hour postinfection
    MPO
    myeloperoxidase
    OPG
    osteoprotegerin
    PMN
    polymorphonuclear leukocyte
    q-PCR
    quantitative PCR
    qRT-PCR
    quantitative RT-PCR
    rm
    recombinant mouse.

  • Received June 28, 2019.
  • Accepted October 30, 2019.

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