Leveraging the Microbiome to Alleviate Ocular Surface Disease

keratic precipitate

Researchers at the University of Pittsburgh are leveraging the microbiome to alleviate ocular surface diseases. This exciting work was the topic of the Eye & Ear Foundation’s May 31st webinar, “Leveraging the Microbiome to Alleviate Ocular Surface Disease.”

Anthony St. Leger, PhD, Assistant Professor in the Departments of Ophthalmology and Immunology, began the presentation. His research interests include the ocular microbiome and its effects on ocular disease. “Most people on this call know that the visual system, like most systems in the body, relies on a series of parts acting in concert to preserve function,” he said. “So, in terms of the visual system, light passes through the optically clear cornea focused by the lens captured by the retina and processed by the brain. My lab is primarily focused on the cornea because if light can’t pass through the cornea, all these downstream events can’t really happen.”

Diseases at the Ocular Surface

Indeed, diseases of the ocular surface (cornea and conjunctiva) can severely affect eyesight and quality of life and include allergies, chemical burns, microbial infections, dry eye Disease/ Sjögren’s syndrome. Current medical treatments include lubricants, steroids, antivirals/antibiotics, and corneal transplants.

While these treatments are relatively effective, there are some disadvantages, specifically with lubricants. They have to be applied multiple times a day, making them laborious. This fix is also temporary as it basically treats a symptom without curing the disease.

Steroids can calm infections at the ocular surface but have their own problems because they suppress the immune system. Using them long-term can result in a higher risk of glaucoma. Antivirals and antibiotics have resistance concerns. Corneal transplants are one of the most well accepted grafts in the human body. “However, my lab and others have shown that if you do a corneal transplant on a hot bed of nerves infected with virus or other diseases, that those grafts are actually more highly rejected,” Dr. St. Leger said. “The overarching goal in my lab is to understand how we can manipulate the ocular surface immune response to alleviate disease.”


The microbiome is a hot topic frequently used in the news today. It is the collection of all microbes, such as bacteria, fungi, viruses, and their genes, that naturally live on our bodies and inside us. Each person is associated with 10-100 trillion microbes. The ratio of human to bacteria is about 1:1.

Microbiota composition is found in different regions: respiratory, oral, skin, gut, vagina. What’s lacking is there is no ocular microbiome here, and that is because the ocular field of ocular microbiome research is just sort of getting going.

When thinking about colonizing microbes, things to consider are the environment, types of nutrients, temperature, and levels of oxygen.

Looking at characteristics of the intestinal immune environment and the ocular immune environment, there are many similarities.

The intestine has mucus continually washing the epithelial layer of the intestine, resident immune cells within and beneath the epithelial layer of the intestine, high concentrations of anti-microbials, antibodies, and other immune factors, and goblet (mucus-producing) cells associated with the tissue. In comparison, the ocular surface has tears continually washing the epithelial layer of the cornea, resident immune cells within and beneath the epithelial layer of the conjunctiva (different for the cornea), high concentrations of anti-microbials, antibodies, and other immune factors, and goblet (mucus-producing) cells associated with the tissue.

Ocular Microbiome

Ocular swabs routinely test negative for bacteria. Sequencing (DNA/RNA) for bacterial signatures began in 2009 and has continued to increase in popularity as time has gone on. Quantification of load reveals that the eye harbors 150-200-fold fewer bacteria per human cell compared to other sites. DNA studies reveal one bacterium per 20 epithelial cells. These studies, however, do not reveal whether bacteria are alive/dead/contaminants/real, etc.

A series of serendipitous findings catapulted Dr. St. Leger’s lab to microbiome research. They had mice lacking the immune factor that controlled bacterial infection. These mice spontaneously developed bacterial infections of the conjunctiva or eyelids – one hint of bacteria. When the lab did sequencing analyses of the ocular surface, they found DNA was present. They ground up a bunch of ocular surface tissue for mice and found one bacterium grew out, which spurred all the studies going forward.

“How do we know which microbes matter?” Dr. St. Leger asked. “We start by identifying if bacteria matter.”

Bacterial dysbiosis is linked to keratitis and uveitis. Antibiotics/germ free status is linked to less uveitis disease. SigA production and release rely on the microbiome.

These studies implicate a role for the microbiome, but they do not identify: 1) whether the relevant bacteria are systemic (gut) or local (ocular surface), 2) a single microbe that’s responsible for physiological responses, 3) whether these microbes affect physiology.

Implicating a role for the microbiome in ocular health means:

  • Immune cells from the intestine (or other peripheral sites) are activated and migrate to ocular surface
  • Soluble factors produced by bacteria (butyrate-de Paiva) can regulate inflammation in the periphery like the eye
  • Gut bacteria can translocate to the circulation and be deposited at the ocular surface for subsequent immune responses
  • Local bacteria can directly stimulate local immunity
  • Local bacteria can produce soluble factors to regulate inflammation

In terms of the microbiome, Dr. St. Leger’s lab is focused on the role(s) that ocular bacteria have on ocular health.

Eye-Colonizing Microbe

An eye-colonizing microbe can protect the ocular surface from disease. Dr. St. Leger’s lab focuses on a bacteria called corynebacterium mastidis (or C. mast), a known colonizer of ocular mucosa. Through a series of downstream immune responses, it protects the ocular surface from infection.

Mice were treated with topical tobramycin (commonly prescribed for P. aeruginosa infection) for a week and then assessed whether ocular surface immunity was affected. Topical antibiotics temporarily eliminates C. mast, but in four weeks, it returns. While antibiotics are disrupting the immune response to the point where ethe eye is more likely to be infected by a blinding pathogen, Dr. St. Leger emphasized that antibiotics are a good thing. He is just suggesting a more judicious approach towards using them.

C. mast is relevant to humans. It stimulates immune cells from the humans. The presence of an immune response suggests that healthy humans have been exposed to this microbe for long enough to generate immunity. The next step is engineering eye-colonizing bacteria to act as a long-term delivery system.

“We asked the question, can we genetically modify C. mast to produce immune regulatory cytokines or produce factors that create more efficient tears to alleviate ocular surface disease?” Dr. St. Leger said. “We’re talking about engineering the colonizing microbe to alleviate this ocular surface disease.”

They chose Interleukin-10 (IL-10) because it suppresses inflammation and disease and enhances wound healing. They have produced three IL-10 candidates and found this genetically engineered microbe can still colonize the eye.

In the most important experiment: if mice are colonized with PBS or wild type C. mast, we see sort of this linear progression of healing, Dr. St. Leger explained. However, if they are colonized with IL-10 producing bacteria, there is a 50% increase in the speed that a wound heals, suggesting that these microbes are actually speeding up wound healing and helping disease. “We’ve neutralized IL-10 in these mice to show that the speed is abrogated, suggesting this is a direct effect of IL-10 and not from some peripheral effect we haven’t thought of yet,” he added.

Dr. St. Leger’s conclusions for Part I:

  • The microbiome does influence ocular (surface) disease
  • Our laboratory is active in using mouse models to identify causal relationships between bacteria and physiology
  • We have engineered an eye-colonizing microbe to secrete a factor to enhance wound healing
  • We are pursuing standards that allow us and future generations effectively study the human microbiome


The second part of the webinar was presented by Dr. Marie Hélène Errera, MD, Pharma D, PhD, Associate Professor of Ophthalmology. She specializes in uveitis and medical retina and surgical diseases. Her research interests include ocular inflammation, immunology, and the epidemiology of uveitis.

Uveitis is inflammation of the eye. It is diagnosed in the clinic with the use of special lamps that reveal inflammatory deposits behind the cornea when the uveitis is in the anterior segment of the eye, which is called anterior uveitis. But the inflammation can affect the middle of the eye (intermediate uveitis), or the back of the eye (posterior uveitis).

With uveitis, there are many diagnoses to consider. Among them are:

  • Idiopathic, no associated disease (around 1/3)
  • Juvenile idiopathic arthritis (children)
  • HLA B27, a genetic factor that increases the risk of developing anterior uveitis. It has a distinct clinical phenotype and may be associated with severe intraocular inflammation and systemic inflammatory diseases, particularly ankylosing spondylitis and reactive arthritis
  • Spondylarthropathy, inflammatory bowel disease, psoriasic arthropathy, rheumatoid arthritis
  • Pars planitis/intermediate uveitis
  • Infections (Herpes virus, cytomegalovirus, toxoplasmosis, Lyme disease…)
  • TINU, which is also an inflammation of the kidneys
  • Sarcoidosis, an inflammatory condition that affects other organs of the body, especially the lungs

Dr. Errera talked about some of these diagnoses. Sarcoidosis, for example, is bilateral and chronic. It has skin, joint, eye involvement, typical lung disease, and lymph nodes. Ocular manifestations are choroidal granuloma and peripheral multifocal choroiditis (inflammation of the choroid behind the retina in the back of the eye) and conjunctival granulomas (inflammatory nodules), keratitis (inflammation of the cornea), retinitis (inflammation of the retina), glaucoma, and involvement of the eyelids and lacrimal glands.

Pars planitis is common, with “snowbanks” or “snowballs” – vitreous condensations without any underlying systemic disease or associated infection. Common complications are cataract, macular edema, vitreous haze, increase in IOP, and retinal detachment.

Another cause of uveitis is HLA B27+. This has a sudden onset with limited duration, with recurrences in 50-75%. Spondylitis occurs in 50% of cases, and uveitis may be the presenting sign of the disease.

The most common system disease associated with pediatric uveitis is juvenile idiopathic arthritis (JIA), in which uveitis develops in about 11-30% and is chronic anterior uveitis. There is often a delay in diagnosis and chronic active inflammation. In up to 15%, there are ocular complications like visual impairment. Glaucoma and cataract can also result.

Posterior uveitis is very rare in the United States, but very frequent in Japan and China. It is known as Vogt-Koyanagi-Harada.


Dr. Errera will be conducting a study of individuals with various causes of uveitis to identify microbial and lifestyle exposures associated with the disease. The gut microbiome will be evaluated during follow up at baseline and in treatment. Approval by the local ethics committee has finally been granted, so the study is about to start with the help of the Microbiome Center at the University of Pittsburgh.

Participants with various causes and types of uveitis of diverse backgrounds (race, gender, ethnicity, and geography) will be asked to provide stool (for microbiome), aqueous fluid and blood samples (for immunophenotyping and hormone levels), and a complete diet history, along with psychological and medication questionnaires.

The goal is to identify if there is a gut microbiome taxonomic and/or functional signature at baseline associated with inflammation, and to identify microbiome associations with cytokines, uveitis treatment (e.g. steroids/immunosuppressive use), lifestyle (diet, smoking, physical activity, obesity, anxiety, sleep, depression, pain), and circulating hormone levels to see if they are associated with uveitis occurrence.

“We will see with future studies if we can adjust or modify treatment – probiotics, dietary change, antibiotics, intervention – depending on the microbiome,” Dr. Errera said.