ABOVE: Illustration of the NLRP3 inflammasome © ISTOCK.COM, SELVANEGRA

New research into the neural mechanisms behind depression concludes that a protein complex called an inflammasome, which induces inflammation and often triggers cell death, may be a key player in the condition, at least in mice.

The study, published in Cell Reports on October 25, probed the mechanisms producing depressive behaviors in a common mouse model in which animals are subjected to chronic mild stress. A combination of in vitro and mouse experiments showed that showed that, when activated by stress, NLRP3 inflammasomes found within immune cells in the brain trigger a so-called neurotoxic response in neighboring cells, eventually leading to the death of nearby neurons. That neurotoxic response is already a well-established contributor to depressive behavior in animals, and separately, the NLRP3 inflammasome been linked in previous research to several human diseases, including Alzheimer’s disease, type 2 diabetes, cardiovascular disease, a rare condition called Muckle-Wells syndrome, and severe COVID-19. The study authors write in their paper that the newly uncovered mechanism could be a novel therapeutic target in depression.

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The study’s authors, who did not respond to interview requests from The Scientist, note in their paper that researchers had previously linked the neurotoxic astrocytes to neuron death in degenerative diseases such as Alzheimer’s and Parkinson’s, and that brain inflammation is known to contribute to depression. Yet little work had probed whether and how neurotoxic astrocytes might play a role in depression.

First, the research team, led by Gang Hu of the Nanjing University of Chinese Medicine, set out to determine whether these astrocytes played a role in their depression model. Two to three times per day for six weeks, the team subjected some groups of mice to stressors, which ranged from hours of physical restraint and full days of starvation to having their cages tilted at 45° angles. As expected, the stressed mice engaged in behaviors akin to depression, such as immobility and reduced interest in rewards. The researchers took brain sections of some stressed and some unstressed mice two and four weeks into the study, allowing them to monitor the progression of stress’s effects on the brain.

Fluorescent imaging studies revealed that chronic stress activates what’s known as the NF-κB pathway, already known to regulate NLRP3 inflammasome activity, in microglia found in the mouse hippocampus. Once activated, the inflammasomes upregulate the gene for caspase-1, an enzyme that triggers the production and release of three cytokines from the microglia: TNF-α, IL-1α, and C1qA. When the researchers cultured primary astrocytes isolated from stressed and unstressed mice and treated the cells with a cocktail of those three cytokines, the astrocytes from stressed mice displayed gene expression changes characteristic of the neurotoxic state. The researchers conclude that inflammasome activity in microglia explains the observed neuronal degradation and depression-like behavioral changes in the mice.

“It’s not surprising to me that the activation [of NLRP3 inflammasomes] leads to the release of these cytokines, and that can in turn activate this other cell type,” says Gloria Lopez-Castejon, an immunology researcher at the University of Manchester who didn’t participate in the new study.

The downstream effects of inflammasome activity

The finding that inflammasomes within one cell type trigger activity in another in this mouse model aligns with what researchers have already learned about inflammasome signaling. Lopez-Castejon says that in vitro studies show that cells tend to die after their inflammasomes are activated, likely as a defense mechanism against pathogens, so other cells are needed to carry on the neurotoxic response. However, she notes that it’s less clear whether this self-destruct mechanism occurs in vivo as well.

The real frontier now is to know how, when, and where inflammasome[s are] activated in humans.

—Pablo Pelegrin, Murcia BioHealth Research Institute-Hospital

To confirm which cells play which roles in the neuroinflammatory pathway, the researchers conducted a series of in vivo and in vitro knockout experiments. The in vitro experiments used cells taken from mice rather than cell lines—a detail that, according to Lopez-Castejon, likely made the in vitro model more accurate. Inhibiting NLRP3 activity in microglia reduced the induction of neurotoxicity in astrocytes and subsequent depression-like behavior in stressed mice. No such reduction was observed when blocking NLRP3 activity in astrocytes, further indicating that microglia play a key role in the signaling pathway identified by the paper.

By selectively removing NLRP3 from one type of cell at a time, “I think they show very nicely that it plays a role in these cells,” says Oregon Health and Science University School of Medicine inflammation researcher Isabella Rauch.

However, Rauch notes that the paper didn’t identify the initial trigger of the pathway—the way in which stress kicks off the chain of events remains unknown. “If we knew what turned it on, that would be even better,” she says, clarifying that there are several such unanswered questions in the inflammasome field and that it’s not troubling or surprising that this paper didn’t provide all the answers at once.

Rauch also points out that the knockout mice used in the study have previously been shown to have developmental behavioral differences compared to wildtype mice, adding that she would have liked to see the researchers address that. She speculates that it’s possible that microglia react differently to stress because of developmental differences rather than due to a lack of NLRP3, and adds that an ideal version of the knockout experiment would involve inhibiting NLRP3 activity only once the mice are grown rather than having it in place for life. “Then you can draw a specific conclusion that it’s playing a role in the developed brain,” she says.

Inflammasomes in translation

Due to NLRP3’s known association with human disease, researchers are currently working to devise inhibitors that would block the inflammatory cascade the protein complex triggers. The researchers in the new study treated stressed mice with one such inhibitor—MCC950, which was developed in 2015 and is being explored in clinical trials—to reduce NLRP3 expression in microglia. They report that MCC950 dampened the induction of neurotoxic astrocytes, alleviating neuroinflammation and depression-like behaviors, just as their NLRP3 gene knockout experiments had. The authors’ success in using an inhibitor to block inflammasome activity “opens the field for pharmacological treatments,” says Rauch.

See “Retrotransposon RNA Triggers NLRC4 Inflammasome Formation: Study”

Despite preliminary indicators that inhibiting inflammasome activity may offer clinical benefits for some conditions, researchers who spoke to The Scientist note that it’s far too early to say with any certainty that doing so could treat or prevent depression in humans.

Lopez-Castejon commends the researchers for the effort they made to “combine all of these [behavioral] test for every single knockout, every experiment. I think that’s good in the sense that they check every time that the model is working.” But as far as human relevance goes, “I don’t know what aspects of human depression this model reflects,” she says, noting that the study examined induced, depression-like behavioral changes in mice rather than depression in humans.

“It’s a starting point,” Pablo Pelegrin, an inflammasome researcher at Murcia BioHealth Research Institute-Hospital in Spain, who didn’t work on the study, writes in an email to The Scientist. “The real frontier now is to know how, when, and where inflammasome[s are] activated in humans,” he says. Pelegrin is the founder of a spinoff company called Viva In Vitro Diagnostics that’s focused on bridging that gap.

The researchers behind the study are upfront about several of these limitations in their paper, especially the imperfections of using stressed animals and cells to study a human psychological condition. But given how the “mechanisms underlying depression are poorly understood,” they argue that their findings, while incomplete, provide insights that “may assist in the development of antidepressants.”