ABOVE: A specialized camera photographed the back of a healthy human eye, including the retina. ©istock, olaaf

The retina is layered with photoreceptors, a variety of other neurons, and protective and structural membranes, but retinal disorders can tamper with this delicate system. Mutations in the Crumbs homolog 1 (CRB1) gene disrupt the integrity of retinal membranes, leading to the gradual loss of photoreceptors. CRB1 mutations associate with multiple retinal diseases, including Leber congenital amaurosis and retinitis pigmentosa. However, since each patient’s disease presents differently, with various types, numbers, and locations of retinal lesions, some scientists suspect that environmental factors interact with the CRB1 gene to influence how disease manifests.

A new study published in Cell demonstrated one way that the environment modifies the phenotype of mice with a CRB1 mutation.1 Researchers showed that CRB1 degradation triggers both a leaky colon epithelial barrier and a leaky retinal pigment epithelium (RPE) barrier in the mice, allowing bacteria to pass from the gut into the bloodstream, and then into the eye, damaging the retina.

Converting photons into electrical signals requires an intricate retinal anatomy. The retina’s inner limiting membrane separates the vitreous cavity of the eye from the axons of the first layer of neurons. Deeper in, the outer limiting membrane, the membrane compromised in CRB1 mutants, divides photoreceptor nuclei from their light-absorbing outer and inner segments. Finally, the RPE separates photoreceptor outer segments from a tissue called the choroid, which has high blood flow.

"The gut part is a new finding,” said Peter Quinn, a Columbia University retinal disease researcher not involved in this study. “The finding that the retinal pigment epithelium is also broken down is a completely new finding.” 

The study authors Lai Wei, a retinal researcher at Sun Yat-sen University, and Richard Lee, a physician-scientist now at the National Eye Institute who studies inflammatory eye disease, hypothesized that an infectious trigger—bacteria or viruses—caused or exacerbated some eye diseases. They wanted to closely examine the eye for bacteria, so they extracted intraocular fluid from the eyes of patients undergoing cataract surgery and analyzed it using shotgun metagenomic sequencing. In 2021, they published a paper in Cell Discovery showing that there was, in fact, an array of bacterial species but no viruses or fungi present in patients’ eyes.2 The species differed depending on whether the patients also had other eye diseases, such as glaucoma or age-related macular degeneration.

Wei and Lee wondered where the bacteria came from. The choroid, the part of the eye with very high blood flow, sits next to the RPE and the rest of the retina; the researchers thought that perhaps during aging, a small amount of bacteria entered the eye through this tissue. “As a first step to simply test the principle of sparse bacterial entry to the eye from the choroidal circulation…we used the Rd8 CRB1 mutant mouse,” wrote Lee in an email. They knew that in patients with CRB1-associated inherited eye disease, the outer retina, the part of the eye next to the choroid, was often abnormal. They hypothesized that this could be because bacteria had entered the bloodstream and were leaking from the choroid blood vessels into the retina. This could cause an immune response that damaged the tissue.

Scientists already knew a good deal about mice with CRB1 mutations. Since CRB1 is a transmembrane protein that helps maintain epithelial barriers and assists in the normal development of photoreceptors, mice with mutations have retinal lesions, among other eye problems. To see if bacteria played a role in the retinal lesions, Wei raised mice with mutated CRB1 in germ-free conditions and discovered that these mice had normal retinas. “We knew we were on to something,” said Lee. 

The team decided to look for bacteria in the retinal lesions of CRB1 mutant mice housed in a non-sterile environment. Using metatranscriptomic analysis, they found low levels of seven bacterial species in the retinal lesions of mutant mice. By using fluorescence in situ hybridization and transmission electron microscopy, the team found bacteria in the lesions but not in the normal retinal tissue in these mutant mice.

To determine where the bacteria came from and how they moved from the bloodstream into the retina, the team used immunostaining. They found that in normal mice, CRB1 was expressed in the junctions between RPE cells as well as the junctions connecting the epithelial cells of the colon. But in CRB1-mutant mice, there was much less CRB1 protein in these places, and both barriers were leaky. Using fluorescence-labeled permeability assays, the team reported that bacteria leaked from the colon into the bloodstream, traveled to the eye, and moved through the leaky RPE barrier into the retina.

They treated the mutant mice with a broad-spectrum antibiotic cocktail and found fewer and smaller retinal lesions. Then they injected an adeno-associated viral vector containing functional CRB1 into the lower GI tract of micethis fixed the leaky gut barrier, but not the retinal barrier. This intervention restored the integrity of the gut barrier, and while the retinal barrier remained leaky, bacteria did not translocate to the eye, and there were far fewer retinal lesions. “You can protect the eye by restoring the gut,” said Lee. “That, for me, is … the killer experiment.”

Quinn cautioned that fewer lesions “does not mean that they prevented retinal degeneration, which is a separate thing.” He said that the lesions in the mutant mice are typical of retinal dysplasia, which may or may not eventually lead to the progressive loss of photoreceptors during aging. Lee countered with a 2011 study in which loss of photoreceptors and retinal degeneration only happen in areas of the retina with lesions.3

The researchers only examined young mice, and Quinn noted that it takes a long time for degeneration to occur. He’d like the researchers to test retinal function in older mice by using an optokinetic tracking reflex test, or pupillometry, and also determine if function improves after antibiotic therapy.

“We don’t know how much this will translate to humans,” said Quinn. “[It’s] not good for the patients to think that this is some sort of magical treatment that they could have.”

The team is planning a human study on patients with CRB1-associated retinal dystrophy. They hope to determine whether the patients’ gut epithelial barriers are intact or leaky, as in the mutant mice. “If we don’t find that the epithelial barrier is abnormal in patients, then this doesn’t translate,” said Lee. However, if it is leaky in humans as well, Lee hopes that treatments could someday help patients with retinal eye diseases exacerbated by bacteria, including “gene therapies of the gut, which could be a one-off big treatment that may be something to consider. And then there are all sorts of slow-release antibiotic implants into the eye that are feasible.”

References

  1. Peng S, et al. CRB1-associated retinal degeneration is dependent on bacterial translocation from the gut. Cell. 2024;187(6):1387-1401.
  2. Deng Y, et al. Identification of an intraocular microbiota. Cell Discovery. 2021;7(13)
  3. Aleman T, et al. Human CRB1-associated retinal degeneration: Comparison with the rd8 Crb1-mutant mouse model, Invest Ophthalmol Vis Sci. 2011;52:6898-6910.