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Published on: August 4, 2023
TLR9 ligand CpG-ODN applied to the injured mouse cornea elicits retinal inflammation
Holly R Chinnery1, Samuel McLenachan, Nicolette Binz
1Department of Anatomy and Developmental Biology, School of Biomedical Sciences, Monash University, Clayton, VIC, Australia.
Abstract:
During bacterial and viral infections, unmethylated CpG-DNA released by proliferating and dying microbes is recognized by toll-like receptor (TLR) 9 in host cells, initiating innate immune responses. Many corneal infections occur secondary to epithelial breaches and represent a major cause of vision impairment and blindness globally. To mimic this clinical situation, we investigated mechanisms of TLR9 ligand-induced corneal inflammation in mice after epithelial debridement. Application of CpG oligodeoxynucleotides (ODNs) resulted in neutrophil and macrophage infiltration to the cornea and loss of transparency. By 6 hours after CpG-ODN administration, TLR9 mRNA was increased in the cornea and retina. In vivo clinical examination at 24 hours revealed inflammatory infiltrates in the vitreous and retina, which were confirmed ex vivo to be neutrophils and macrophages, along with activated resident microglia. CpG-ODN-induced intraocular inflammation was abrogated in TLR9(-/-) and macrophage-depleted mice. Bone marrow reconstitution of irradiated TLR9(-/-) mice with TLR9(+/+) bone marrow led to restored corneal inflammatory responses to CpG-ODN. Fluorescein isothiocyanate-CpG-ODN rapidly penetrated the cornea and ocular media to reach the retina, where it was present within CD68(+) retinal macrophages and microglia. These data show that topically applied CpG-ODN induces intraocular inflammation owing to TLR9 activation of monocyte-lineage cells. These novel findings indicate that microbial CpG-DNA released during bacterial and/or viral keratitis can cause widespread inflammation within the eye, including the retina.
Insights
Microbial CpG-DNA triggers toll-like receptor 9 (TLR9) in the eye, causing inflammation in the cornea and retina. This immune response, mediated by macrophages, highlights a potential mechanism for vision impairment during infections.
Area of Science:
- Immunology
- Ophthalmology
- Microbiology
Background:
- Corneal infections, often following epithelial damage, are a leading cause of vision loss.
- Unmethylated CpG-DNA from microbes activates toll-like receptor 9 (TLR9), initiating innate immune responses.
- The role of TLR9 in ocular inflammation following epithelial breaches is not fully understood.
Purpose of the Study:
- To investigate the mechanisms of TLR9 ligand-induced corneal and intraocular inflammation after epithelial debridement.
- To determine the cellular and molecular pathways involved in CpG-oligodeoxynucleotide (ODN)-induced ocular inflammation.
Main Methods:
- Induction of corneal epithelial debridement in mice followed by topical application of CpG-ODNs.
- Assessment of inflammatory cell infiltration (neutrophils, macrophages) and ocular transparency.
- Analysis of TLR9 mRNA expression in ocular tissues.
- Evaluation of intraocular inflammation in the vitreous and retina.
- Utilizing TLR9-deficient mice and macrophage depletion models.
- Bone marrow reconstitution experiments.
- Tracking of fluorescein isothiocyanate-labeled CpG-ODN distribution within the eye.
Main Results:
- Topical CpG-ODN application induced significant neutrophil and macrophage infiltration into the cornea, leading to loss of transparency.
- TLR9 mRNA levels increased in the cornea and retina within 6 hours.
- Inflammatory infiltrates, including neutrophils, macrophages, and activated microglia, were observed in the vitreous and retina.
- CpG-ODN-induced intraocular inflammation was dependent on TLR9 and macrophages.
- CpG-ODN rapidly penetrated the cornea and ocular media to reach retinal macrophages and microglia.
- Bone marrow reconstitution restored corneal inflammatory responses in TLR9-deficient mice.
Conclusions:
- Topically applied CpG-ODN induces intraocular inflammation via TLR9 activation of monocyte-lineage cells.
- Microbial CpG-DNA released during bacterial or viral keratitis can lead to widespread ocular inflammation, including the retina.
- These findings provide insights into the pathogenesis of infectious keratitis and associated vision impairment.
