Pattern recognition receptor research yields promising results in keratitis

By Laird Harrison

New research into pattern-recognition receptors is yielding promising therapies that target the specific pathogens and inflammatory pathways in keratitis, researchers said.

“We have reached an exciting point in the study of infectious keratitis, with so many molecular pathways being identified,” they wrote. The researchers, from three centres in Southampton, UK, published a review of these findings in the journal Eye.

Corneal infection or inflammation causes about 1% of visual impairment worldwide, the researchers wrote, citing World Health Organisation figures.

In the United Kingdom, the condition hospitalises 4000 people per year. Trauma caused by agrarian activity is the major cause, and studies in the United States have correlated an increase in infections with the use of contact lenses, the researcher found.

Although the intact corneal epithelium is a formidable barrier, it can be breached, for example by epithelial abrasion, the researchers wrote. In the case of contact lenses, they speculate that biofilms on the lens surfaces may give the pathogens a relatively safe harbour where they can adapt to the corneal defences.

Once they have breached the epithelium, the pathogens may directly damage the cornea. The inflammatory response and the treatments used to suppress the infections may also cause damage leading to blindness.

The researchers divided pathogens infecting the cornea into five categories: viruses, bacteria, fungi, parasites and nematodes. Recent research has revealed much about how immune cells detect these pathogens and respond to them.

Pattern-recognition receptors (PRRs) such as Toll-like receptors (TLRs) and NOD- (nucleotide-binding oligomerisation domains) like receptors (NLRs), play a crucial role in recognizing pathogen-associated molecular patterns. Recognition of these patterns stimulates a spiralling inflammatory response, the authors reported. They described the immune response to various pathogens:

When pseudomonas aeruginosa, the most common cause of Gram-negative bacterial keratitis, breaches the corneal epithelium, TLR4 and TLR5 on macrophages in the corneal stroma recognise its lipopolysaccharide and flagellin molecules. They initiate inflammation, recruiting neutrophils and macrophages, which results in corneal haze. And they cause the release of antimicrobial factors such as nitric oxide.

TLR2 recognises another common cause of keratitis, Staphylococus aureus, by identifying bacterial lipoproteins, peptidoglycan molecules and S. aureus protein A. Another unknown receptor appears to recognize S. aureus protein as well. As part of the resulting inflammatory response, epithelial cells release β-defensin 2.

Responses to infection by Onchocerca volvulus (onchocerciasis) varies from one individual to another. This nematode maintains an endosymbiotic relationship with Wolbachia bacteria. Previous exposure to antigens appears to be necessary to cause a systemic response. TLR2 and TLR6 recognize Wolbachia products, activating T-cells, macrophages and neutrophils and later eosinophils. The neutrophils may cause the progressive corneal haze seen in onchocerciasis.

Fungal spores are able to penetrate compromised epithelium where they germinate. Macrophage Dectin-1 and Dectin-2 receptors recognise the β-glucan and α-mannan molecules on their surfaces, which then recruit neutrophils as part of an inflammatory response that eventually also involves T-cells.

Herpes simplex virus (HSV), thought to be the primary infectious cause of blindness, stimulates multiple immune responses. Experiments in mice have shown that TLR3 and TLR9 recognise membrane glycoproteins that the virus uses to bind to the host cells. TLR4 identifies endogenous heat shock protein 70 and β-defensin-3 expressed by corneal cells in response to the virus.

And human corneal epithelial cells have been shown to respond to HSV1 via TLR3 and to express TLR7. Activation of these pathways recruits dendritic cells, macrophages, neutrophils, natural killer cells and T-cells.

A ubiquitous protozoan, Acanthamoeba can severely and persistently infect the cornea. It can encyst and reactivate after treatment ends. Infection does not protect against reinfection, suggesting that the innate immune system may be the cornea’s primary defence. ImmunoglobulinA inhibits Acanthamoeba from binding to epithelial cells, complements activation and opsonisation and augments neutrophil killing.

As a tophozoite, Acanthamoeba attaches to the cell surface via mannose-binding protein. It releases a mannose-induced serine protease which causes cytolysis of corneal epithelial cells, enabling it to infiltrate deeper layers of the corneal tissue.

TLR4 recognises Acanthamoeba and releases pro-inflammatory and chemotactic mediators. Macrophages and neutrophils can kill Acanthamoeba including cysts, but the cysts do not give off chemotactic stimulus that can be recognised.

Steroid treatment increases the virulence of the disease. The protozoan can maintain endosymbiotic relationships with bacteria that increase corneal toxicity and can confound the diagnosis.

Such findings may lead to new therapeutic targets, the authors reported. They offered the following summary:

Clinicians often rely on topical steroids. This non-specific attempt to reduce inflammation can cause complications such as corneal thinning, perforation and increased intra-ocular pressure, resulting in worse outcomes, particularly in Acanthamoeba, Norcardia and HSV1. However, the Steroids for Corneal Ulcers Trial showed that steroids have no significant benefit or harm when used as an adjuvant to treatments of bacterial keratitis in the short term and appear to improve visual outcomes at 12 months post treatment.

More tailored therapies under development include inhibiting ataxia telangiectasia mutated kinase. In a mouse model of HSV1, this reduced the severity keratitis. In human cultured corneas, it slowed viral replication.

A high-affinity human monoclonal antibody to S. aureusα-toxin reduces corneal damage. A new antibiotic, targocil, has also shown preliminary success in inhibiting staphylococcal infections.

Lithium chloride, and a capase-1 inhibitor that reduces the amount of IL-1β produced, may improve Pseudomonas infection.

The apoptotic Fas pathway, which regulates the production of pro-inflammatory cytokines, and a phosphor-inositol-3-kinase/Akt pathway that activates a triggering receptor on myeloid cells 2, offer avenues to reduce inflammation.

In fungal keratitis, small interfering RNA targeted against TLR2 improved the outcome of A. fumigatus infection in a rat model. Decreased fungal burden and reduced production of pro-inflammatory cytokines led to clearer corneas and fewer instances of perforation.

Heat shock protein HSPB4 appears to act as a damage-associated molecular pattern activating TLR2 and the NF-kB pathway. Inhibition of this pathway, via HSPB4 antibodies or TNF-α stimulated gene/protein 6, suppressed macrophage activation and resultant neutrophil infiltration, leading to an improvement in corneal clarity.

Doxycycline may depelete Wolbachia bacteria, facilitating treatment of O. volvulus worms.

Il-1β monoclonal antibody kanakinumab appeared to help in a case report of cryopyrin-associated periodic fever syndrome. Likewise, anakira, an IL-1 antagonist, has successfully treated corneal infiltrates.

Collagen cross-linking, using ultraviolet-A light and riboflavin, creates covalent bonds to cross-link collagen fibres in the corneal stroma, increasing the strength of the cornea and halting the corneal melting process caused by a variety of microbes. The approach has shown more promise in bacterial and Acanthamoeba than fungal infections, however. In the end, it is possible it could halt drug penetration.

The researchers speculate that collagen cross-linking in combination with anti-inflammatory agents based on pathogen-associated molecular patterns, newer antimicrobials and anti-matrix metallo-proteinases, will most effectively preserve infected corneal tissue.

 “We may now be closer than previously imagined to effective, targeted therapies to treat corneal tissue damage and subsequent blindness as a result of the global disease that is infectious keratitis,” the authors concluded.