November 2005
Volume 46, Issue 11
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Immunology and Microbiology  |   November 2005
Silencing Toll-like Receptor-9 in Pseudomonas aeruginosa Keratitis
Author Affiliations
  • Xi Huang
    From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
  • Ronald P. Barrett
    From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
  • Sharon A. McClellan
    From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
  • Linda D. Hazlett
    From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.
Investigative Ophthalmology & Visual Science November 2005, Vol.46, 4209-4216. doi:10.1167/iovs.05-0185
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      Xi Huang, Ronald P. Barrett, Sharon A. McClellan, Linda D. Hazlett; Silencing Toll-like Receptor-9 in Pseudomonas aeruginosa Keratitis. Invest. Ophthalmol. Vis. Sci. 2005;46(11):4209-4216. doi: 10.1167/iovs.05-0185.

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      © 2015 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To determine the effects of silencing Toll-like receptor (TLR) 9 signaling in Pseudomonas aeruginosa keratitis.

methods. Corneal TLR9 mRNA levels were tested by RT-PCR in C57BL/6 (B6, susceptible) and BALB/c (resistant) mice and compared. The response of B6 mice to CpG DNA, which binds TLR9, was tested after subconjunctival injection of mice with control or CpG DNA; TLR9, IL-1β, macrophage inflammatory protein (MIP)-2, IL-4, IL-10, IL-12, IL-18, and IFN-γ levels were measured by RT-PCR. Langerhans cells (LCs) were stimulated with CpG DNA and treated with TLR9 or control siRNA, and mRNA levels of TLR9, IL-1β, and MIP-2 were detected by RT-PCR. In addition, IL-1β levels were tested by ELISA. Then B6 mice were injected subconjunctivally with control or TLR9 siRNA before infection and treated topically afterward. Slit lamp, clinical score, RT-PCR, ELISA, myeloperoxidase assay, and plate counts were performed.

results. TLR9 mRNA levels were sixfold higher in B6 than in BALB/c corneas the day after injection. B6 mice injected with CpG DNA exhibited an increase in corneal mRNA for TLR9, IL-1β, MIP-2, IL-12, and IFN-γ over controls. LCs stimulated with CpG DNA and treated with TLR9 siRNA exhibited reduced TLR9, IL-1β, and MIP-2 levels compared with controls. Finally, B6 mice treated with TLR9 siRNA showed decreases in corneal opacity, polymorphonuclear leukocyte number, IL-12 and IFN-γ mRNA, IL-1β, and MIP-2 protein compared with those treated with control siRNA. Fewer corneas perforated in these mice, but bacterial loads were higher than in controls.

conclusions. Signaling through TLR9 appears important in P. aeruginosa keratitis, and silencing TLR9 signaling reduces inflammation but likely contributes to decreased bacterial killing in the cornea.

Toll-like receptors (TLRs) are key components of the innate immune system that detect microbial infection and trigger antimicrobial host responses. 1 2 Gram-positive and Gram-negative bacteria activate TLRs and induce the secretion of proinflammatory molecules, mainly chemokines and cytokines, including, but not limited to, interleukin (IL)-1β, macrophage inflammatory protein (MIP)-2, and tumor necrosis factor (TNF)-α, which amplify the response to infection. If unregulated, these inflammatory molecules also contribute to the pathologic effects induced by many other bacteria, including Pseudomonas aeruginosa. 3 4  
P. aeruginosa keratitis is one of the most common and destructive of bacterial diseases, with a high incidence in extended-wear contact lens users. 5 Experimental animal studies have shed some light on mechanisms involved in the development of keratitis 6 and have shown that dominant T-helper type 1 (TH1) responder mouse strains (e.g., B6) are susceptible (cornea perforates), whereas dominant TH2 responder strains (e.g., BALB/c) are resistant (cornea heals) after similar bacterial challenge. 7 8 These well-defined in vivo animal models provide a unique opportunity to test whether corneal expression levels of members of the TLR superfamily are important in mediating innate and directing divergent-adaptive immune responses resulting in phenotype differences (susceptibility vs resistance) after P. aeruginosa ocular challenge. 
Several studies have examined the role of TLRs in vitro in human corneal epithelial cells 9 10 and in vivo in knockout mice and lipopolysaccharide (LPS)- or TLR-specific ligand challenge, 11 but no studies have tested the role of TLRs in bacteria-induced keratitis. We chose to focus our study on TLR9 because it recognizes microbial DNA characterized by an abundance of unmethylated CpG dinucleotides 12 13 that induce a strong TH1 inflammatory response. 14 Moreover, TLR9 already has been found likely to have an important function in the corneal epithelium and to signal through the common adaptor molecule, myeloid differentiation factor-88 (MyD88) on CpG DNA challenge. 11 In contrast to other TLR agonists, CpG DNA is superior in activating dendritic cells (DCs) and inducing costimulatory molecules and proinflammatory cytokines. 15 During infection, recognition of CpG DNA from intracellular pathogens skews and fine-tunes the ongoing immune response and induces a long-lasting TH1 milieu. 16 17 In the cornea of B6 mice, the end result of a TH1-mediated response to P. aeruginosa infection is devastating and results in corneal perforation. 7 8 Thus, testing methods to downregulate or redirect a TH1 response in B6 mice appeared attractive as an approach to modulate disease and to understand the role of TLR9 in pathogenesis. 
In this regard, RNA interference (RNAi) is a naturally occurring biologic process for silencing alleles during development in plants, 18 invertebrates, and vertebrates, in which the presence of double-stranded RNA (dsRNA) in a cell results in the destruction of mRNAs whose sequences share homology with the dsRNA. 19 These processes represent an evolutionarily conserved system to protect against viral and bacterial infection and the genomic instability caused by transposable elements and repetitive sequences. 20 In mammalian cell culture, 21 reduction in gene expression also has been accomplished by transfecting synthetic RNA oligonucleotides. These RNAs are termed small-interfering (si) RNAs; siRNA has been shown to specifically degrade homologous RNA in mammalian cells. 22 23  
The therapeutic potential of siRNA has been demonstrated in several models of disease, including viral infection, 24 25 26 sepsis, 27 and ocular neovascularization. 28 29 These successes prompted us to explore the feasibility of siRNA treatment in a model of P. aeruginosa keratitis in which corneal perforation is the outcome. In this study we report that silencing TLR9 signaling using TLR9 siRNA works in principle because it successfully downregulated the expression of important early inflammatory cytokines such as IL-1β and MIP-2 in cultured LC. We also show that siRNA knockdown of mouse TLR9 in vivo reduced the host inflammatory response, evidenced by decreased proinflammatory cytokines, fewer polymorphonuclear leukocytes (PMNs) infiltrating the cornea, and fewer perforated corneas in B6 mice infected with P. aeruginosa. Despite these beneficial effects, bacterial load was increased, suggesting the likely important role of TLR9 in bacterial clearance in the infected cornea. 
Methods
Infection
Eight-week-old female B6 and BALB/c mice (n = 5/group per time; The Jackson Laboratory, Bar Harbor, ME) were anesthetized with ether and placed beneath a stereoscopic microscope at 40 × magnification, and the cornea of the left eye was wounded with three 1-mm incisions using a sterile 25-gauge needle. A bacterial suspension (5 μL) containing 1 × 106 colony-forming units (CFUs) of P. aeruginosa ATCC strain 19660, prepared as described, 8 was applied to the eye surface. Eyes were examined 24 hours after infection or at other times described here to ensure that mice were similarly infected and to monitor disease. B6 mice (n = 5 per group per treatment) were injected (20 μg/mouse) subconjunctivally with CpG or GpC (control) DNA. The GpC DNA (control) and CpG DNA were designed with a GAGCTT and a GACGTT motif, respectively. B6 mice (n = 5 per group per treatment) were infected as described after subconjunctival injection and topical treatment with TLR9 or control siRNA. Animals were treated humanely and in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Ocular Response
Corneal disease was graded as described 8 : 0 = clear or slight opacity, partially or fully covering the pupil; +1 = slight opacity, fully covering the anterior segment; +2 = dense opacity, partially or fully covering the pupil; +3 = dense opacity, covering the entire anterior segment; and +4 = corneal perforation or phthisis. A clinical score was calculated after in vivo infection to express disease severity, and slit lamp photography illustrated the disease response. 
Real-Time Reverse Transcription–Polymerase Chain Reaction
Methods for real-time RT-PCR have been described by others. 30 In this study, mouse corneas or LCs were homogenized in RNA (STAT-60; Tel-Test; Friendsville, TX), and total RNA was isolated according to the manufacturer’s instructions to produce a cDNA template for PCR reaction. Primers used were 5′-AGCTCAACCTGT CCTTCAATTACCGC-3′ (sense) and 5′-ATGCCGTTCATGTTCAGCTCCTGC-3′ (antisense) for mouse TLR9; 5′-CGCAGCAGCACATCAACAAGAGC-3′ (sense) and 5′-TGTCCTCATCCTGGAAGGTCC ACG-3′ (antisense) for mouse IL-1β; 5′-TGTCAATGCCTGAAGACCCTGCC-3′ (sense) and 5′-AACTTTTTGACCGCCCTTGAGAGTGG-3′ (antisense) for mouse MIP-2; 5′-TGT CAT CCT GCT CTT CTT TCT CG-3′ (sense) and 5′-GTT TGG CAC ATC CAT CTC CG-3′ (antisense) for mouse IL-4; 5′-TGCTAACCGACTCCTTAATGCAGGAC-3′ (sense) and 5′-CCTTGATTTCTG GGCCATGCTTCTC-3′ (antisense) for mouse IL-10; 5′-GGT CAC ACT GGA CCA AAG GGA CTA TG-3′ (sense) and 5′-ATT CTG CTG CCG TGC TTC CAA C-3′ (antisense) for mouse IL-12-p40; 5′-GCC ATG TCA GAA GAC TCT TGC GTC-3′ (sense) and 5′-GTA CAG TGA AGT CGG CCA AAG TTG TC-3′ (antisense) for mouse IL-18; 5′-CAGAGCCAGATTATCTCTTTCTACCTC AGAC-3′ (sense) and 5′-CTTTTTCGCCTTGCTGTTGCTGAAG-3′ (antisense) for mouse IFN-γ; and 5′-GATTACTGCTCTGGCTCCTAGC-3′ (sense) and 5′-GACTCATCGTACTCCTGCTTGC-3′ (antisense) for mouse β-actin. For PCR amplification, 1 μL each cDNA sample was used for each 25-μL PCR reaction. Real-time measurements were analyzed in duplicate in three independent runs (Cepheid Smart Cycler System; Cepheid Inc., Sunnyvale, CA). Relative mRNA levels were calculated after normalizing to β-actin. 31  
LC Culture
Mouse LCs (XS52 cell line derived from BALB/c skin) were routinely cultured in complete RPMI (with 10% fetal calf serum [FCS]) supplemented with 0.5 ng/mL murine recombinant granulocyte macrophage–colony-stimulating factor (GM-CSF) and 5% fibroblast (NS line) supernatant. LCs were stimulated with CpG DNA and treated with TLR9 or control siRNA. Fluorescent dye (DAPI; Roche Diagnostics, Mannheim, Germany) that binds selectively to DNA was used as a nuclear marker (staining was performed according to the manufacturer’s protocol). 
RNA Interference
siRNA duplexes targeting the coding region of TLR9 were produced (Silence siRNA Cocktail Kit; Ambion Inc., Austin, TX). After RNase III digestion of dsRNA and siRNA purification, siRNA targeting TLR9 was obtained, tested, and used as described in “siRNA Delivery.” Control siRNA was similarly produced from a scrambled DNA sequence. 
siRNA Delivery
LCs were seeded into 6-well plates at a density of 1.0 × 105 cells/well in normal growth medium. After 36 hours, 60 nM TLR9 siRNA was used to form an siRNA complex (siPORT; Ambion). Control siRNA was similarly treated. The TLR9 or control siRNA complex was added to LCs, and this was followed by 16 hours of incubation under routine culture conditions. Next, 2 mL fresh normal growth medium was added to each well. LCs were stimulated with 3 μg/mL rhodamine (Rho)–conjugated CpG DNA (Rho-CpG) for 6 hours. Cell lysates were prepared for total RNA extraction, and supernatant was collected for ELISA assay. In vivo use of siRNA has been described by others. 28 For the studies described herein, TLR9 or control siRNA was injected (10 μL/4 μM per mouse) subconjunctivally into the left eye of B6 mice 1 day before infection and then topically onto the infected cornea three times (5 μL/4 μM per mouse) for 48 hours after infection. 
ELISA
Protein levels were quantitated using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN). LC culture supernatants were collected and tested for IL-1β after CpG DNA stimulation and after TLR9 or control siRNA treatment. In vivo, after TLR9 or control siRNA treatment, individual corneas (n = 5/group per time) were harvested from infected mice at 5 days after infection and analyzed for IL-1β and MIP-2 protein levels. Samples were homogenized with a glass pestle (Kontes; Fischer, Itasca, IL) in PBS with 0.1% Tween 20, centrifuged at 10,000g, and assayed per the manufacturer’s instruction. 
Immunostaining
Corneas from TLR9 siRNA– or control siRNA–treated B6 mice (Jackson Laboratory) were removed (n = 3/group) at 5 days after infection, snap frozen in optimum temperature cutting (OCT) compound, and stored at –20°C. Ten-micrometer–thick sections were cut, dried overnight at 37°C, and incubated with anti-TLR9 monoclonal antibody (mAb; clone 5G5; 1:20 dilution), followed by a biotinylated secondary antibody (1:250) each for 1 hour and by staining visualized using diaminobenzidine (DAB). Controls were treated similarly, but the primary antibody was omitted. Sections were photographed with a microscope (Axiophot; Zeiss, Oberkochen, Germany) equipped with a digital camera system (Axiocam; Zeiss). Six hundred inflammatory cells per group were counted, and the percentages of TLR9-positive cells were calculated. 
Quantitation of PMN
Samples were assayed for myeloperoxidase (MPO) activity, as described. 32 Corneas from TLR9 siRNA– or control siRNA–treated mice (n = 5/group) were collected 5 days after infection and homogenized in 1.0 mL of 50 mM phosphate buffer, pH 6.0, containing 0.5% HTAB. Samples were freeze-thawed three times and centrifuged at 14,000g for 10 minutes Supernatant (0.1 mL) was added to 2.9 mL of the 50 mM phosphate buffer containing o-dianisidine dihydrochloride (16.7 mg/100 mL) and hydrogen peroxide (0.0005%). The change in absorbance at 460 nm was monitored for 5 minutes (Helios-α; Thermo Spectronics, Rochester, NY), and the results were expressed as units of MPO per cornea (1 U MPO activity = ∼2.0 × 105 PMN). 
Bacterial Load
Bacteria were quantitated in the infected corneas of B6 mice after TLR9 or control siRNA treatment. Corneas (n = 5 per group) were collected 5 days after infection Each cornea was homogenized in sterile 0.9% saline containing 0.25% BSA. Serial tenfold dilutions of the samples were plated on Pseudomonas isolation agar (Difco, Detroit, MI) in triplicate, and plates were incubated overnight at 37°C. Results are reported as log10 number of CFU per cornea. 
Statistical Analysis
Unpaired, two-tailed Student’s t-test was used to determine statistical significance of real-time RT-PCR, ELISA, cell count, clinical score, MPO, and bacterial plate count. Data were considered significant at P < 0.05. All experiments were repeated at least twice to ensure reproducibility, and representative data from a single experiment are shown. 
Results
TLR9 Signaling
Using well-defined experimental P. aeruginosa keratitis models (B6 versus BALB/c), 8 we first tested for TLR9 mRNA expression in the infected cornea. Data (Fig. 1A)showed a 30-fold and a 5-fold increase (6-fold difference) in mRNA for TLR9 in the B6 and BALB/c corneas, respectively, 1 day after infection. To specifically test TLR9 signaling in vivo, CpG or GpC (control) DNA was injected subconjunctivally into the left eye of B6 mice. TLR9 mRNA expression was significantly increased approximately 6-fold compared with control levels 5 hours after injection. IL-1β, MIP-2, and the TH1-type cytokines IL-12, IL-18, and IFN-γ were used as measures of corneal inflammation, and IL-4 and IL-10 TH2-type cytokines were similarly tested. mRNA levels compared with controls were enhanced 38-fold (IL-1β), 48-fold (MIP-2), 5-fold (IL-12), and 2.5-fold (IFN-γ), respectively (Figs. 1B 1C) . Levels of IL-4 and IL-10 mRNA were unchanged over control levels (Fig. 1D)
Specific Blocking of TLR9 Signaling in LC by siRNA
LCs were used to develop and test siRNA techniques before in vivo use. Before CpG DNA stimulation, LCs were transfected with either TLR9 or control siRNA. To trigger TLR9 signaling, LCs were exposed to CpG DNA or GpC DNA (control) 36 hours after transfection. Figure 2comparatively shows that Rho CpG–stimulated, control siRNA–treated cells (Fig. 2C)appeared similar to the Rho CpG DNA only–stimulated positive control cells (Fig. 2B) , whereas the TLR9 siRNA plus Rho CpG–treated cells (Fig. 2D)appeared similar to unstimulated DAPI-stained LCs (Fig. 2A) . To further confirm TLR9 silencing, mRNA expression levels of TLR9 and cytokines IL-1β and MIP-2 were tested and found reduced over controls by 80%, 75%, and 85%, respectively (Table 1) . To confirm these data, the reduction of IL-1β protein was selectively measured with ELISA (Fig. 3)and was decreased over controls approximately 70% after TLR9 siRNA treatment. These results clearly demonstrated the ability of TLR9 siRNA to downregulate TLR9 expression in vitro and subsequently to inhibit TLR9 signaling. 
Reduction of Ocular Inflammation by TLR9 siRNA
After demonstrating the feasibility of siRNA delivery to LCs, significantly suppressing endogenous TLR9 expression and reducing IL-1β and MIP-2 mRNA and IL-1β protein in vitro, we next silenced TLR9 in vivo in the B6 mouse cornea. To confirm that TLR9 siRNA was effectively maintained in the cornea, TLR9 immunostaining (Figs. 4A 4B)was used after treatment with control siRNA (Fig. 4A)or TLR9 siRNA (Fig. 4B) . After gene silencing, reduced expression of TLR9 was evident until 5 days after infection (only time tested) in the epithelium and in infiltrating cells. TLR9-positive cells were counted, and a reduced number (P < 0.001) of positive cells (600 cells counted per group) were detected (Fig. 4C)in the TLR9 siRNA–treated versus the control siRNA–treated cornea. 
B6 mice were subconjunctivally injected with control or TLR9 siRNA, and ocular disease was observed and graded. Five days after infection, 4 of 5 corneas had perforated in the control group (in 2 independent experiments); therefore, the experiments were terminated. In a separate experiment (data not shown) when animals were observed 7 days after infection, the data were no different from those shown 5 days after infection. Five days after infection, slit lamp (Figs. 5A 5B)and clinical score (Fig. 5C)showed less opacity and disease, and fewer corneas perforated in the TLR9 siRNA– than in the control siRNA–treated group (P = 0.014). mRNA levels for IL-12, IL-18, and IFN-γ (Fig. 5D)and IL-4 and IL-10 (Fig. 5E) , prototypical of TH1 and TH2 cytokines, respectively, also were measured at 5 days after infection using RT-PCR. IL-12 and IFN-γ mRNA levels were reduced significantly (P = 0.014 and P = 0.021, respectively), whereas IL-18, IL-4, and IL-10 levels were unchanged in the corneas of TLR9 siRNA– compared with control siRNA–treated mice (Figs. 5D 5E) . Moreover, at this time, ELISA (Figs. 6A 6B)of B6 mouse corneas treated with TLR9 siRNA compared with control siRNA showed reduced protein levels for IL-1β (P = 0.015) and MIP-2 (P = 0.001) and a reduced number of PMNs (Fig. 6C)(fourfold; P = 0.0002). In contrast, bacterial load (Fig. 6D)was increased (about 0.5 log) in TLR9 siRNA mice compared with control siRNA–treated mice (P = 0.016). 
Discussion
Innate immunity provides a first line of host defense against bacterial growth and spread in the early phase of infection. Critical to the innate immune response are cells such as PMN, macrophages, and DCs. 33 Recent studies demonstrate that innate immunity provides for recognition of conserved pathogen-associated molecular patterns (PAMPs) through TLR/IL-1R expressed on the cell surfaces of various cell types, including immune cells. 34 Recognition of invading pathogens may trigger cytokine/chemokine production and the upregulation of costimulatory molecules in phagocytes. This may lead to elimination of the pathogen directly or through the activation of cells of the acquired arm of the immune response. 
One member of the TLR superfamily, TLR9, recognizes bacterial DNA, characterized by an abundance of unmethylated CpG dinucleotides, which distinguishes bacterial from mammalian DNA. Mammalian DNA has a low frequency of CpG dinucleotides that are mostly methylated and have low immunostimulatory activity. In contrast to other TLR agonists, CpG DNA is superior in the activation of DCs and the induction of costimulatory molecules (e.g., CD80, CD86) and cytokines such as interleukin (IL)-12 and IL-18. This qualifies CpG DNA as a TH1-promoting adjuvant, capable of fine-tuning the ongoing immune response and inducing a long-lasting TH1 milieu. 35 36 In the eyes of B6 mice, the end result of a TH1-mediated response to experimental P. aeruginosa infection is devastating, resulting in corneal perforation, whereas a TH2-dominant response, as in BALB/c mice, results in less disease and healing. 7 8 In the present study, we have focused on TLR9 for the reasons given and because others have shown that CpG DNA treatment of B6 wild-type (wt) and TLR9 knockout mice to activate TLR9 leads to the development of sterile keratitis in wt, but not knockout, mice. 37 To begin our study, RT-PCR was used and revealed that TLR9 levels were increased sixfold in the infected corneas of B6 compared with BALB/c mice. The specific role of TLR9 in disease was also confirmed by injections of B6 mice with CpG DNA that binds TLR9, and this resulted in significant (early) fold increases in corneal mRNA levels of TLR9, IL-1β, MIP-2, IL-12, and IFN-γ. In contrast, IL-4, IL-10, and IL-18 mRNA levels were unchanged. 
These data suggested that the downregulation of TLR9 signaling by siRNA treatment might reduce the innate response sufficiently to cause less host damage in the infected corneas of B6 mice. In this regard, several proof-of-principle experiments already have demonstrated the therapeutic potential of siRNA. siRNA protected mice from fulminant hepatitis, 22 38 viral infection, 25 sepsis, 27 and the ocular neovascularization that causes macular degeneration. 28  
In other models, mice endogenously lacking MyD88 demonstrated an impaired clearance of P. aeruginosa from the lung, with little PMN recruitment. In addition, these mice exhibited severe deficiency in MIP-2, the chemokine that recruits PMN into inflammatory sites, 39 showing similarity to our studies silencing only TLR9. By selectively silencing TLR9 rather than MyD88, through which most of the TLRs signal 1 40 41 we hypothesized that it would be possible that other TLRs that use this pathway would be sufficient to remove the bacterial pathogen and at the same time induce less corneal bystander damage. The host side of this prediction was correct in that protein levels for the cytokine IL-1β and the chemokine MIP-2, and mRNA for IL-12 and IFN-γ—all proinflammatory and shown of importance in P. aeruginosa infection in other studies 7 42 43 —were downregulated. However, in the absence of TLR9, bacterial load was elevated (0.5 log), and PMN numbers were reduced significantly, indicating the importance of TLR9 in bacterial clearance and pathogenesis. 
The significant decrease in PMN number probably accounts for the deficient bacterial killing after TLR9 treatment. This cell is the first immune cell to arrive at the site of infection. PMNs quickly initiate microbicidal functions, including the production of antimicrobial products and proinflammatory cytokines 44 that serve to contain the infection. They are of critical importance in microbial keratitis because depletion of these cells using an anti-PMN antibody (RB6 to 8C5) in inbred B6 or BALB/c mice before ocular infection with P. aeruginosa resulted in death in a significant percentage of mice. 45 PMNs detect the presence of a pathogen through germline-encoded receptors that recognize microbe-associated molecular patterns; the best-characterized in vertebrates was TLR. All the TLRs (TLR1–10) except TLR3 are expressed in human PMNs and in human cells. 44 In human PMNs, the response to the TLR9 agonist CpG DNA required GM-CSF pretreatment, something not tested in this study. 
In summary, we have provided evidence that in P. aeruginosa keratitis, TLR9 siRNA treatment works in principle to reduce excess inflammation by the downregulation of proinflammatory cytokines and cellular infiltrate. In addition, we have shown that bacterial load in the cornea was greater in TLR9 siRNA–treated mice than in control siRNA–treated mice, indicating that TLR9 signaling is likely critical in cytokine/chemokine regulation, PMN infiltration, and bacterial clearance. 
 
Figure 1.
 
(A) TLR9 mRNA expression in BALB/c and B6 normal and infected cornea 1 day after infection. (BD) RT-PCR of mRNA expression levels (normalized to β-actin) of TLR9, IL-1β, MIP-2, IL-12, IL-18, IFN-γ, IL-4, and IL-10 in CpG or GpC (control) DNA–treated B6 mouse cornea 5 hours after treatment. p.i., postinfection.
Figure 1.
 
(A) TLR9 mRNA expression in BALB/c and B6 normal and infected cornea 1 day after infection. (BD) RT-PCR of mRNA expression levels (normalized to β-actin) of TLR9, IL-1β, MIP-2, IL-12, IL-18, IFN-γ, IL-4, and IL-10 in CpG or GpC (control) DNA–treated B6 mouse cornea 5 hours after treatment. p.i., postinfection.
Figure 2.
 
Detection of TLR9 protein in LC (nuclei stained blue with DAPI). (A) Negative control shows nuclei (blue) of non-CpG–treated LCs. (B) Positive control uses Rho-CpG; red indicates interaction with TLR9 on LCs. (C) Control siRNA– and Rho-CpG–treated LCs appear similar to those in B (positive control). (D) TLR9 siRNA–treated LCs show reduced Rho-CpG interaction (less red staining) for TLR9. Magnification, ×100.
Figure 2.
 
Detection of TLR9 protein in LC (nuclei stained blue with DAPI). (A) Negative control shows nuclei (blue) of non-CpG–treated LCs. (B) Positive control uses Rho-CpG; red indicates interaction with TLR9 on LCs. (C) Control siRNA– and Rho-CpG–treated LCs appear similar to those in B (positive control). (D) TLR9 siRNA–treated LCs show reduced Rho-CpG interaction (less red staining) for TLR9. Magnification, ×100.
Table 1.
 
Real-time PCR of Fold Changes (Normalized to β-actin) in TLR9, IL-1β, and MIP-2 mRNA Expression in XS52 LC Treated with TLR9 siRNA and CpG DNA
Table 1.
 
Real-time PCR of Fold Changes (Normalized to β-actin) in TLR9, IL-1β, and MIP-2 mRNA Expression in XS52 LC Treated with TLR9 siRNA and CpG DNA
Treatment TLR9 IL-1β MIP-2
CpG only (control) 7.8 225.6 237.5
Control siRNA + CpG 5.1 148.2 157.1
TLR9 siRNA + CpG 1.4 53.5 41.2
Figure 3.
 
IL-1β protein levels were significantly (P < 0.01 for each concentration tested) decreased in TLR9 siRNA–treated compared with control siRNA–treated, CpG DNA–stimulated LCs.
Figure 3.
 
IL-1β protein levels were significantly (P < 0.01 for each concentration tested) decreased in TLR9 siRNA–treated compared with control siRNA–treated, CpG DNA–stimulated LCs.
Figure 4.
 
TLR9 immunostaining in cornea after treatment with control siRNA (A) or TLR9 siRNA (B). After gene silencing, reduced expression of TLR9 is evident 5 days after infection in the epithelium and on infiltrated cells, showing that silencing is effective. Magnification, ×40. (C) Percentage of TLR9-positive cells in control siRNA–treated and TLR9 siRNA–treated cornea. A significantly reduced number of TLR9-positive cells (P < 0.001) was detected below controls in the TLR9 siRNA–treated cornea. Six hundred cells per group were counted.
Figure 4.
 
TLR9 immunostaining in cornea after treatment with control siRNA (A) or TLR9 siRNA (B). After gene silencing, reduced expression of TLR9 is evident 5 days after infection in the epithelium and on infiltrated cells, showing that silencing is effective. Magnification, ×40. (C) Percentage of TLR9-positive cells in control siRNA–treated and TLR9 siRNA–treated cornea. A significantly reduced number of TLR9-positive cells (P < 0.001) was detected below controls in the TLR9 siRNA–treated cornea. Six hundred cells per group were counted.
Figure 5.
 
Slit lamp (AB) and clinical score (C) of ocular disease response in B6 (susceptible) mice treated with TLR9 siRNA or control siRNA. Perforation is evident in the control siRNA–treated eye (B), whereas the eye of TLR9 siRNA–treated mice (A) shows a +3 (dense opacity over anterior segment) response (AB; magnification, ×40). Clinical score (C) showed less disease and fewer corneas perforated in the TLR9 siRNA– versus the control siRNA–treated group (P = 0.014 at 5 days after infection). IL-12 and IFN-γ mRNA levels (D) were reduced significantly (P = 0.014; P = 0.021), whereas IL-18, IL-4, and IL-10 mRNA levels (DE) were unchanged in the corneas of TLR9 siRNA–and control siRNA–treated mice 5 days after infection.
Figure 5.
 
Slit lamp (AB) and clinical score (C) of ocular disease response in B6 (susceptible) mice treated with TLR9 siRNA or control siRNA. Perforation is evident in the control siRNA–treated eye (B), whereas the eye of TLR9 siRNA–treated mice (A) shows a +3 (dense opacity over anterior segment) response (AB; magnification, ×40). Clinical score (C) showed less disease and fewer corneas perforated in the TLR9 siRNA– versus the control siRNA–treated group (P = 0.014 at 5 days after infection). IL-12 and IFN-γ mRNA levels (D) were reduced significantly (P = 0.014; P = 0.021), whereas IL-18, IL-4, and IL-10 mRNA levels (DE) were unchanged in the corneas of TLR9 siRNA–and control siRNA–treated mice 5 days after infection.
Figure 6.
 
ELISA assay of B6 mouse cornea treated with TLR9 siRNA versus control siRNA (AB). Reduction in the level of (A) IL-1β (2.5-fold; P = 0.015) and (B) MIP-2 (<2-fold; P = 0.001) is significant for each. (C) Corneal MPO activity and (D) bacterial plate count in TLR9 siRNA– versus control siRNA–treated B6 mice after PA challenge. (C) A significantly reduced (4-fold) number of PMNs was detected in the corneas of TLR9 siRNA– versus control siRNA–treated mice 5 days after infection (P = 0.0002). (D) Bacterial load 5 days after infection was significantly increased (about 0.5 log) after TLR9 siRNA versus control siRNA treatment (P = 0.016).
Figure 6.
 
ELISA assay of B6 mouse cornea treated with TLR9 siRNA versus control siRNA (AB). Reduction in the level of (A) IL-1β (2.5-fold; P = 0.015) and (B) MIP-2 (<2-fold; P = 0.001) is significant for each. (C) Corneal MPO activity and (D) bacterial plate count in TLR9 siRNA– versus control siRNA–treated B6 mice after PA challenge. (C) A significantly reduced (4-fold) number of PMNs was detected in the corneas of TLR9 siRNA– versus control siRNA–treated mice 5 days after infection (P = 0.0002). (D) Bacterial load 5 days after infection was significantly increased (about 0.5 log) after TLR9 siRNA versus control siRNA treatment (P = 0.016).
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Figure 1.
 
(A) TLR9 mRNA expression in BALB/c and B6 normal and infected cornea 1 day after infection. (BD) RT-PCR of mRNA expression levels (normalized to β-actin) of TLR9, IL-1β, MIP-2, IL-12, IL-18, IFN-γ, IL-4, and IL-10 in CpG or GpC (control) DNA–treated B6 mouse cornea 5 hours after treatment. p.i., postinfection.
Figure 1.
 
(A) TLR9 mRNA expression in BALB/c and B6 normal and infected cornea 1 day after infection. (BD) RT-PCR of mRNA expression levels (normalized to β-actin) of TLR9, IL-1β, MIP-2, IL-12, IL-18, IFN-γ, IL-4, and IL-10 in CpG or GpC (control) DNA–treated B6 mouse cornea 5 hours after treatment. p.i., postinfection.
Figure 2.
 
Detection of TLR9 protein in LC (nuclei stained blue with DAPI). (A) Negative control shows nuclei (blue) of non-CpG–treated LCs. (B) Positive control uses Rho-CpG; red indicates interaction with TLR9 on LCs. (C) Control siRNA– and Rho-CpG–treated LCs appear similar to those in B (positive control). (D) TLR9 siRNA–treated LCs show reduced Rho-CpG interaction (less red staining) for TLR9. Magnification, ×100.
Figure 2.
 
Detection of TLR9 protein in LC (nuclei stained blue with DAPI). (A) Negative control shows nuclei (blue) of non-CpG–treated LCs. (B) Positive control uses Rho-CpG; red indicates interaction with TLR9 on LCs. (C) Control siRNA– and Rho-CpG–treated LCs appear similar to those in B (positive control). (D) TLR9 siRNA–treated LCs show reduced Rho-CpG interaction (less red staining) for TLR9. Magnification, ×100.
Figure 3.
 
IL-1β protein levels were significantly (P < 0.01 for each concentration tested) decreased in TLR9 siRNA–treated compared with control siRNA–treated, CpG DNA–stimulated LCs.
Figure 3.
 
IL-1β protein levels were significantly (P < 0.01 for each concentration tested) decreased in TLR9 siRNA–treated compared with control siRNA–treated, CpG DNA–stimulated LCs.
Figure 4.
 
TLR9 immunostaining in cornea after treatment with control siRNA (A) or TLR9 siRNA (B). After gene silencing, reduced expression of TLR9 is evident 5 days after infection in the epithelium and on infiltrated cells, showing that silencing is effective. Magnification, ×40. (C) Percentage of TLR9-positive cells in control siRNA–treated and TLR9 siRNA–treated cornea. A significantly reduced number of TLR9-positive cells (P < 0.001) was detected below controls in the TLR9 siRNA–treated cornea. Six hundred cells per group were counted.
Figure 4.
 
TLR9 immunostaining in cornea after treatment with control siRNA (A) or TLR9 siRNA (B). After gene silencing, reduced expression of TLR9 is evident 5 days after infection in the epithelium and on infiltrated cells, showing that silencing is effective. Magnification, ×40. (C) Percentage of TLR9-positive cells in control siRNA–treated and TLR9 siRNA–treated cornea. A significantly reduced number of TLR9-positive cells (P < 0.001) was detected below controls in the TLR9 siRNA–treated cornea. Six hundred cells per group were counted.
Figure 5.
 
Slit lamp (AB) and clinical score (C) of ocular disease response in B6 (susceptible) mice treated with TLR9 siRNA or control siRNA. Perforation is evident in the control siRNA–treated eye (B), whereas the eye of TLR9 siRNA–treated mice (A) shows a +3 (dense opacity over anterior segment) response (AB; magnification, ×40). Clinical score (C) showed less disease and fewer corneas perforated in the TLR9 siRNA– versus the control siRNA–treated group (P = 0.014 at 5 days after infection). IL-12 and IFN-γ mRNA levels (D) were reduced significantly (P = 0.014; P = 0.021), whereas IL-18, IL-4, and IL-10 mRNA levels (DE) were unchanged in the corneas of TLR9 siRNA–and control siRNA–treated mice 5 days after infection.
Figure 5.
 
Slit lamp (AB) and clinical score (C) of ocular disease response in B6 (susceptible) mice treated with TLR9 siRNA or control siRNA. Perforation is evident in the control siRNA–treated eye (B), whereas the eye of TLR9 siRNA–treated mice (A) shows a +3 (dense opacity over anterior segment) response (AB; magnification, ×40). Clinical score (C) showed less disease and fewer corneas perforated in the TLR9 siRNA– versus the control siRNA–treated group (P = 0.014 at 5 days after infection). IL-12 and IFN-γ mRNA levels (D) were reduced significantly (P = 0.014; P = 0.021), whereas IL-18, IL-4, and IL-10 mRNA levels (DE) were unchanged in the corneas of TLR9 siRNA–and control siRNA–treated mice 5 days after infection.
Figure 6.
 
ELISA assay of B6 mouse cornea treated with TLR9 siRNA versus control siRNA (AB). Reduction in the level of (A) IL-1β (2.5-fold; P = 0.015) and (B) MIP-2 (<2-fold; P = 0.001) is significant for each. (C) Corneal MPO activity and (D) bacterial plate count in TLR9 siRNA– versus control siRNA–treated B6 mice after PA challenge. (C) A significantly reduced (4-fold) number of PMNs was detected in the corneas of TLR9 siRNA– versus control siRNA–treated mice 5 days after infection (P = 0.0002). (D) Bacterial load 5 days after infection was significantly increased (about 0.5 log) after TLR9 siRNA versus control siRNA treatment (P = 0.016).
Figure 6.
 
ELISA assay of B6 mouse cornea treated with TLR9 siRNA versus control siRNA (AB). Reduction in the level of (A) IL-1β (2.5-fold; P = 0.015) and (B) MIP-2 (<2-fold; P = 0.001) is significant for each. (C) Corneal MPO activity and (D) bacterial plate count in TLR9 siRNA– versus control siRNA–treated B6 mice after PA challenge. (C) A significantly reduced (4-fold) number of PMNs was detected in the corneas of TLR9 siRNA– versus control siRNA–treated mice 5 days after infection (P = 0.0002). (D) Bacterial load 5 days after infection was significantly increased (about 0.5 log) after TLR9 siRNA versus control siRNA treatment (P = 0.016).
Table 1.
 
Real-time PCR of Fold Changes (Normalized to β-actin) in TLR9, IL-1β, and MIP-2 mRNA Expression in XS52 LC Treated with TLR9 siRNA and CpG DNA
Table 1.
 
Real-time PCR of Fold Changes (Normalized to β-actin) in TLR9, IL-1β, and MIP-2 mRNA Expression in XS52 LC Treated with TLR9 siRNA and CpG DNA
Treatment TLR9 IL-1β MIP-2
CpG only (control) 7.8 225.6 237.5
Control siRNA + CpG 5.1 148.2 157.1
TLR9 siRNA + CpG 1.4 53.5 41.2
Copyright 2005 The Association for Research in Vision and Ophthalmology, Inc.
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