Inflammasomes are huge, multi-protein complexes inside cells that spark inflammation when they sense danger. Inflammasomes can also sound the alarm when no pathogens are present, contributing to diseases such as multiple sclerosis. There are many different types of inflammasomes, and the mechanisms behind how some of them form under so-called sterile conditions has been a mystery.
In a new paper published December 3 in Science Immunology, Jayakrishna Ambati of the University of Virginia School of Medicine and his team show that RNA transcribed from a type of mobile genetic element called SINE causes the NLRC4 inflammasome to form when there’s no infection, and for the first time, they reveal a receptor that senses this rogue RNA. Inhibiting NLRC4 inflammasome activation in an animal model of atrophic macular degeneration (AMD) prevented retinal degeneration.
This work “could open up more therapeutic targets for treating dry AMD because now there are a few more players that can be targeted,” says AMD researcher Yvette Wooff of the Australian National University in Canberra who was not involved in the study. But Harvard Medical School ophthalmologist and retina researcher Demetrios Vavvas, also not involved in the study, cautions that “like any study, this will need further replication and validation by an independent group.”
Research into inflammasomes has surged in recent years, and scientists know that in order for an inflammasome to form and then cause inflammation, something has to trigger it. In the case of the NLRC4 inflammasome, during an infection bacterial proteins bind to a protein called NAIP, which recruits and links together several NLRC4 proteins, as well as other proteins called ASC and pro-caspase-1. All of these components together form the NLRC4 inflammasome. Then, pro-caspase-1 is cleaved into caspase-1, which causes the formation and release of inflammatory IL-1β and IL-18 cytokines.
But researchers didn’t know what triggers the formation of the NLRC4 inflammasome when there are no bacterial fragments to bind to NAIP. Ambati’s lab has been interested in the interaction between inflammasomes and SINE (short for short interspersed nuclear element) RNA, which comes from mobile genetic elements known as retrotransposons. These SINEs are only transcribed into RNAs during times of cellular stress, such as an infection or aging. Previous research has found that when too many SINE RNAs hang around the cell, they can contribute to diseases such as Alzheimer’s, systemic lupus erythematosus, AMD, multiple sclerosis, and type 2 diabetes. Researchers knew that these SINE RNAs could induce inflammasome activity, but attempts to identify cytoplasmic RNA sensors that detect SINE RNA and trigger inflammasome formation in the absence of an infection had turned up empty-handed.
Ambati, a cofounder of the company Inflammasome Therapeutics, which is developing a compound that blocks both the NLRP3 and NLRC4 inflammasomes; Shao-bin Wang, then a postdoc in Ambati’s lab; and their colleagues searched for proteins that interact with SINE RNAs. They transfected human cells with SINE RNAs or a control, and purified any SINE RNA complexes that formed. They identified 12 proteins using liquid chromatography-mass spectrometry, five of which were known to be RNA binding proteins. They then targeted each one with siRNAs to see whether taking them out of commission would dampen the release of inflammatory signals known as cytokines, and only siRNA targeting a protein called DDX17 reduced the release of the cytokine IL-1β in SINE RNA–transfected cells. Immunofluorescence in human cells showed clusters of DDX17 and SINE RNAs in the cytoplasm. The team also found that when they depleted DDX17 in cells, they blocked SINE RNA’s activation of caspase-1 (which in turn activates IL-1β) and IL-1β release.
“That’s the surprising thing, that there is a specific sensor for this thing. Certainly it could have been the case that there was no molecular sensor—that it was just causing structural changes or physical changes or ion flux changes that were resulting in this inflammasome being assembled,” says Ambati.
The researchers also determined that this type of NLRC4 inflammasome that forms under noninfectious conditions also includes NLRP3, a protein usually associated with its own eponymous inflammasome. Both the NLRC4 and NLRP3 proteins are necessary to form an active NLRC4 inflammasome when the complex is triggered by SINE RNAs. But interestingly, NLRC4 doesn’t need its normal partner, NAIP. In addition, the researchers found that when DDX17 was unavailable, there was less interaction between the NLRC4 and NLRP3 proteins in SINE RNA-transfected cells. “It was really a surprise that you had this double inflammasome that was being formed,” says Ambati.
The team also examined this system in models of disease. In white blood cells isolated from people with active lupus, the scientists found an increase in SINE RNAs, DDX17 mRNA and protein, and activated caspase-1 compared to healthy controls. Using fluorescence in situ hybridization and proximity ligation assay, they also saw cytosolic DDX17 colocalized with the SINE RNAs in these cells. When they targeted DDX17, NLRP3, or NLRC4 with siRNAs, levels of the cytokine IL-18 fell (IL-1β was undetectable in most of the samples).
In further experiments, the researchers created a mouse model of AMD by injecting SINE RNAs under the retina, which caused retinal pigmented epithelium (RPE) degeneration—a hallmark of AMD—in wildtype mice but not in those engineered to lack NLRC4. Knocking down DDX17 blocked RPE degeneration in mice treated with SINE RNA.
While he says the findings are interesting, Vavvas adds that “the potential implication for diseases like macular degeneration is limited, like all studies are limited, from the imperfect nature of the animal models that we have.” He points out, for example, that mice don’t have maculas, the central part of the retina responsible for sharp vision and the location of AMD. He adds that “another potential conclusion of the study is the protein kinase C delta could also be a target for this type of disease.” In this paper, the researchers show that protein kinase C delta is needed to help form the NLRC4 inflammasome.
Apart from the finding’s potential therapeutic impact, however, Ambati says, “now that we know what is the sensor—at least a sensor—of SINE retrotransposons, that opens up a whole new intersection between RNA biology and immunology.”