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

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.

Figure 1.. PA infection increases IL23/17-axis signaling in B6 mouse cornea.

(A) Mouse corneas were scratched with a needle and inoculated with 1.0 × 10⁴ CFU PA. Whole corneas were collected at 1dpi for quantitative real-time PCR or semiquantitative RT-PCR analysis of IL-23, IL-17A, IL-17RA, IL-17RC. (N=3) (B) Western-blot analysis of IL-23, IL-23R, IL-17A, IL17-RC in cell lysates of whole corneas infected with PA at 1dpi. 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, **p<0.01, ***p<0.001 (One-way ANOVA). (C) The corneas were excised and processed for immunohistochemistry analysis at 1dpi. The 6μm cryostat sections were stained with anti-IL17RA (green), anti-IL23R (green), and DAPI (blue) for nuclei. Two independent experiments were performed; 1 representative image for each condition is presented. E, epithelium; S, stroma. PA, P. aeruginosa. (D) Flow cytometric analyses of IL-17RA and IL-17RC positive immune cells in naïve and infected (Inf) corneas. 10 corneas were pooled for each sample. Percentage of IL17RA, IL17RC and Ly6G positive cells are shown in the flow cytometric plots.

Figure 2.. IL-17RA, IL-17RC neutralizing antibodies decrease the severity of PA infection in B6 mouse cornea.

Mice were subconjunctivally injected with IL-17RA (400ng/5μl), IL-17RC (400ng/5μl) neutralizing antibody 4h before the inoculation with 1.0 × 10⁴ CFU PA. Mouse IgG serves as control. (A) Mouse corneas were monitored and photographed (original magnification × 10) at 1dpi. The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. (B &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<0.05, **p<0.01 (paired t-test).

Figure 3.. IL-17A neutralizing antibody and rmIL-17 have opposing effects on the outcome of PA infection in B6 mouse corneas.

Mice were subconjunctivally injected with IL-17A neutralizing antibody (250ng/5μl) or recombinant mouse IL-17A (200ng/5μl in 0.1% BSA) 4h before the inoculation with 1.0 × 10⁴ CFU PA. Mouse IgG or 0.1% BSA serves as control. (A&D) Mouse corneas were monitored and photographed (original magnification × 10) at 1dpi. The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. (B&E&C&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, C, mean ± SEM). (N=5) *p<0.05, **p<0.01 (paired t-test). (G) Mouse corneas were treated with anti-IL17 antibody, or rmIL-17A and inoculated with P. aeruginosa, Naïve corneas were used as negative Control. The corneas were excised and processed for immunohistochemistry analysis at 1dpi. The 6μm cryostat sections were stained with NIMP-R14 antibody for neutrophils. The images of neutrophils (green) were merged with DAPI (blue nuclei) staining. Two independent experiments were performed; 1 representative image for each condition is presented. E, epithelium; S, stroma. NL, naive cornea.

Figure 4.. Blockade of IL-17A alteres gene expression in B6 mouse corneas in response to PA infection.

Mouse corneas were treated with anti-IL17A antibody or control IgG and inoculated with 1.0 × 10⁴ CFU PA. Corneal epithelial cells 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).

Figure 5.. IL-17A regulated OPG expression in B6 mouse corneas in response to PA infection.

Mouse Corneas were treated with anti-IL17A antibody or rmIL-17A and inoculated with 1.0 × 10⁴ CFU PA. Whole corneas were collected at 1dpi. (A) Protein array analysis revealed the effect of IL-17A on cytokine expression. Selected images for Osteoprotegerin was shown. (B) q-PCR analysis of Osteoprotegerin in whole corneas infected with P. aeruginosa at 1dpi. (N=3) (C) Western-blot analysis of Osteoprotegerin in cell lysate of whole corneas infected with P. aeruginosa at 1dpi. β-actin serves as the loading control. Data are representative of three independent experiments with three corneas per group (B, mean ± SEM). *p<0.05, **p<0.01 (One-way ANOVA). (D) The corneas were excised and processed for immunohistochemistry analysis at 1dpi. The 6μm cryostat corneal sections were stained with anti-Osteoprotegerin (green) and DAPI (blue) for nuclei. Two independent experiments were performed; 1 representative image for each condition is presented. E, epithelium; S, stroma. OPG, Osteoprotegerin.

Figure 6.. Blockade of OPG attenuates the severity of PA infection in B6 mouse cornea.

Mice were subconjunctivally injected with Osteoprotegerin neutralizing antibody (200ng/5μl) 4h before the inoculation with 1.0 × 10⁴ CFU PA. Mouse IgG serves as control. (N=5) (A) Mouse corneas were monitored and photographed (original magnification×10) at 1dpi. The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. (B&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 1dpi. q-PCR analysis of Anti-OPG in whole corneas infected with PA at 1dpi. Data are representative of three independent experiments with three corneas per group (B, C, D mean ± SEM). (N=3) *p<0.05, **p<0.01 (One-way ANOVA).

Figure 7.. OPG upregulation is partially responsible for IL-17-worsened outcome of PA keratitis in B6 mice.

Mouse corneas were treated with rmIL-17A or rmIL-17A+Anti-OPG antibody and inoculated with 1.0 × 10⁴ CFU PA, 0.1% BSA serves as control. (A) Mouse corneas were monitored and photographed (original magnification×10) at 1dpi. The numbers within each eye micrograph are the clinical scores assigned and presented as median plus interquartile range. (B&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, C, mean ± SEM). (N=5) *p<0.05, **p<0.01 (One-way ANOVA).

Figure 8.. Blockade of either IL-17A or OPG attenuates the severity of PA keratitis in B6 mouse cornea at 3 dpi.

Mice were subconjunctivally injected with either IL-17A neutralizing or OPG neutralizing antibody 4h before the inoculation with 1.0 × 10⁴ CFU PA. 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&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.. Blockade of IL-17A, but not OPG, promotes Th2 response to PA infection in B6 mice.

Subconjunctivally injected with either IL-17A neutralizing or OPG neutralizing antibody 4h before the inoculation with 1.0 × 10⁴ CFU PA. Whole corneas were collected at 3dpi, q-PCR analysis of (A) IL23/17 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).

Figure 10.. Concurrent topical application of IL-17A neutralizing antibody and ciprofloxacin eradicates PA-infection associated inflammation in B6 mice.

C57BL/6 mouse corneas were inoculated with 1.0 × 10⁴ CFU PA. Topical solution containing ciprofloxacin (Cip) was used to dissolve IL-17A neutralizing antibody. Topical antibiotic with or without anti-IL17A Ab was then applied, starting 16 hours after infection, at 2-hour intervals during the first and second days of treatment and at 4-hour intervals on the third day of treatment. The infected corneas were (A) photographed and (B) scored, and (C) myeloperoxidase (MPO) activity assay was performed at the end of experiment. The results are representative of 3 independent experiments. Data are representative of three independent experiments with five corneas per group (B, C, mean ± SEM). (N=5) *p<0.05, **p<0.01 (paired t-test).