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NEI-funded Labs Implicate Inflammasomes in Common Eye Disease

AMD destroys the light-sensing eye tissue unit.
Figure 1: AMD Destroys the Light-Sensing Eye Tissue Unit
AMD destroys the retina, retinal pigment epithelium, and choroid. All three help the eye convert light into electrical signals sent to the brain. Drusen are yellow-white fat and protein clusters deposited between the RPE and choroid. Courtesy of the NEI and Washington University School of Medicine Neuroscience Tutorial, St. Louis, MO.

The NLRP3 inflammasome.
Figure 2: The NLRP3 Inflammasome
NLRP3 proteins detect harmful molecules from in or outside of a cell. After detection NLRP3 proteins combine with two other proteins, PYCARD and inactive caspase-1, to form an active NLRP3 inflammasome. The inflammasome activates caspase-1, an enzyme which, in turn, activates interleukins that are secreted into the blood. Traditionally interleukins act like flares, signaling for help from immune cells in combating infections. Courtesy of the NEI and adapted from Davis & Ting, 2010, Nature 11(2) p. 105.

GA RPEs have more NLRP3 inflammasome proteins.
Figure 3: GA RPEs have more NLRP3 Inflammasome proteins
Staining of RPEs from GA (top) and Control (bottom) subjects shows that RPEs have more NLRP3. Tissue donated from deceased GA and aged-matched control subjects were stained with antibodies against NLRP3 (dark blue). Courtesy of Ambati Lab, University of Kentucky.

NLRP3 inflammasome activity may protect eye tissue during wet AMD.
Figure 4: NLRP3 Inflammasome Activity may Protect Eye Tissue during Wet AMD
Blood vessel growth reminiscent of wet AMD was induced in the eyes of transgenic mice that lack NLRP3 (middle) or IL-18 (right) by briefly burning the retinas with a laser. Six days later the retinas were stained with antibodies against isolectin to visualize blood vessels (red). Retinas from the transgenic mice had more blood vessels than control retinas (left) suggesting NLRP3 and IL-18 normally prevent blood vessel growth during wet AMD. Courtesy of O'Neill and Humphries labs, Trinity College Dublin.

The immune system defends our bodies against germs. Every day, immune cells circulate though our bodies, constantly patrolling for infections, such as viruses and bacteria. These cells use molecular warning systems to spot infections and recruit other immune cells to fight them. Recently, two NEI-funded research groups independently discovered that one of the warning systems, called the NLRP3 inflammasome, may be involved in two advanced forms of age-related macular degeneration (AMD), a leading cause of blindness in the U.S. Interestingly, one group found that NLRP3 inflammasomes may damage the eye during one form of AMD whereas another group found they may protect it during another form.

AMD causes about 30 percent of all vision loss in the U.S., usually affecting people over 50. Vision loss is caused by the destruction of three tissue layers lining the inside of the eye called the retina, retinal pigment epithelium (RPE), and choroid (See Figure 1). These tissues normally work together to convert light into electrical signals sent to the brain

'Wet' and 'Dry' describe two basic forms of AMD. Dry AMD is more common, affecting about 90 percent of all patients. Both forms are characterized by the appearance of drusen, which are yellow-white fat and protein clusters deposited in between the RPE and choroid. Wet AMD is further characterized by abnormal blood vessel growth.

Periodic eye injections of drugs that prevent blood vessel growth can improve vision for wet AMD patients and taking certain antioxidant and zinc dietary supplements can reduce the chances some patients will progress to the most severe stages of wet or dry AMD. Nevertheless, studies predicting that the number of people affected by AMD will rise have increased the need for novel effective treatments for all forms of the disease.

Two recent studies, one published in Cell (April 26, 2012) and one in Nature Medicine (April 8, 2012), coincidentally showed for the first time that both forms of AMD may involve the NLRP3 inflammasome and that the molecules associated with it may be effective targets for treating AMD.

Inflammasomes are groups of molecules traditionally found in immune cells. Results from the Cell study, led by Jayakrishna Ambati, M.D. at the University of Kentucky, suggested that NLRP3 inflammasome activation in RPE cells causes tissue destruction during geographic atrophy (GA), a late and severe stage of dry AMD. Whereas results from the Nature Medicine study, led by Drs. Luke A J O'Neill and Peter Humphries at Trinity College in Dublin, Ireland, suggested that NLRP3 inflammasome activation in immune cells protects eye tissue during wet AMD.

Like guards on watch, NLRP3 molecules are proteins found inside cells that search for harmful molecules (See Figure 2). Once they "spot" harmful molecules, NLRP3 proteins "sound the alarm" by recruiting other proteins, called PYCARD and caspase-1, to cluster and form active inflammasomes. Clustering starts a chain of reactions that ultimately cause cells to secrete molecules, called interleukins, or cytokines, into the blood. Interleukins are proteins, traditionally secreted by immune cells, which act like flares, alerting other immune cells in the body of danger.

Previously, it was thought that NLRP3 and other immune cell proteins, such as Toll-like receptors (TLRs), detected only harmful molecules from outside the body, such as bacterial proteins and DNA from viruses. However, more recent studies suggest that during some diseases, such as gout and type-2 diabetes, certain naturally occurring molecules can activate NLRP3 inflammasomes and that drugs targeting interleukins may be effective treatments for these types of diseases. Similarly, results from the Cell and Nature Medicine reports suggested that NLRP3 may recognize harmful molecules found in the eye during AMD.

The Cell report suggested that Alu RNA molecules activate NLRP3 inflammasomes in RPE cells. Alu RNA molecules are short chains of ribonucleic acids (RNA), which regulate genes and are normally cut into small pieces by a protein, called DICER. Previous results from the Ambati lab suggested that tissue destruction in GA was caused by high levels of uncut Alu RNA in RPE cells resulting from low DICER levels.

Initial experiments in the Cell report suggested that only NLRP3 can recognize Alu RNA and activate inflammasomes whereas other immune cell proteins, including TLRs, do not. The researchers then discovered that RPE cells from GA patients had more NLRP3 inflammasome proteins than normal (See Figure 3), suggesting they are involved with GA. These surprising results are the first demonstration of inflammasome molecules in human RPE.

Experiments performed on cultured human RPE cells or on the eyes of transgenic mice suggested that during GA, excess Alu RNA activates NLRP3 inflammasomes in RPE cells causing them to secrete an interleukin called IL-18. Further experiments suggested that IL-18 secretion during GA may cause tissue destruction by inducing cell death in neighboring RPE cells.

In contrast, the Nature Medicine report, suggested that immune cell NLRP3 inflammasomes are involved in other forms of AMD. First, the researchers found that drusen may activate immune cell inflammasomes. Drusen collected from the eyes of AMD patients induced robust IL-1 and IL-18 secretion by human primary blood mononuclear cells (PMBCs), which are immune cells that can access the eye tissues damaged by AMD. Using a similar type of immune cell from transgenic mice, the researchers then showed that proteins found in drusen, such as C1q, may work together to activate NLRP3 inflammasomes causing the immune cells to secrete interleukins.

Then experiments performed on the eyes of transgenic mice suggested that immune cell NLRP3 inflammasomes may be active during wet and dry AMD. Interestingly, these experiments suggested that, unlike GA, activation of immune cell NLRP3 inflammasomes may protect eye tissues during wet AMD by secreting IL-18 (See Figure 4).

Furthermore, treating cultured human RPE cells or mouse brain blood vessel cells with IL-18 reduced secretion of vascular endothelial growth factor (VEGF), a molecule that stimulates blood vessel growth during wet AMD. These results suggested that molecules in the NLRP3 inflammasome pathway may be effective treatment targets for wet AMD.

Currently, researchers are performing clinical trials to test a variety of new AMD treatments, including administering drugs that target the immune system, free-radicals, and growth factors. The preliminary reports described in this article alert the vision research community that novel targets for treating all forms of AMD may also be found in the NLRP3 inflammasome pathways.

- By Christopher G. Thomas, Ph.D.

Publication Citations:

Grant Support:

The studies lead by the Ambati lab (Tarallo et al.) were supported by NIH Grants EY018350, EY018836, EY020672, EY022238, EY019778, EY020442, EY021757, EY021521, EY021721, EY017182, EY017950, EY003040, EY001545, AI063331, AR052756, GM068414, HL091812, & RR033173.

The studies lead by the O'Neill and Humpries labs (Doyle et al.) were supported by NIH Grants EY014240, EY016813, & GM021249.

Last Reviewed: July 2012












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