July 1, 2015 (Vol. 35, No. 13)

Richard A. A. Stein M.D., Ph.D.

A Signaling Nexus for Both Good and Ill, It’s Pathways of Pathogenic and Therapeutic Importance Are Still Being Identified

A recent paradigm shift in medicine started with the finding that several medical conditions, previously not envisioned to have much in common at the molecular level, share sustained inflammatory processes in various organs as a common mechanism of pathogenesis.

Amidst these advances, the inflammasome, a collection of multimeric cytosolic protein complexes, is assuming a central position in understanding pathogenesis and in developing therapeutic strategies.

Several inflammasome assemblies have been described in multiple species, and they all share the same basic architecture, with a sensor molecule, an adaptor, and an effector procaspase. Despite similarities in the proteins that can participate in inflammasome organization, different inflammasome assemblies can be activated by a wide range of signals, including tissue damage and cellular stress, microbial pathogens, inflammation, and metabolic perturbations.

Because the inflammasome is highly adaptable and capable of being activated by many different signals and mechanisms, it can serve as “a flexible scaffold in terms of the immune defense,” says Russell E. Vance, Ph.D., associate professor of immunology and pathogenesis at the University of California, Berkeley.

In the case of canonical inflammasome assemblies, which have been studied more extensively, activation leads to signaling via two pathways. One pathway involves the cleavage and activation of caspase 1, which initiates signaling via the proinflammatory cytokines interleukin-1β and interleukin-18. The other pathway involves the activation of pyroptosis, a proinflammatory form of cell death.

Different inflammasome assemblies can be activated by a wide range of signals, including tissue damage, cellular stress, microbial pathogens, metabolic perturbations, and inflammation, as shown in this X-ray of an elbow. [iStock/David Marchal]

Inflammasome Sensors

Inflammasome sensors can belong to the NOD-like receptor (NLR) or the AIM2-like receptor (ALR) families. The NLR sensors have a tripartite organization and contain an N-terminal effector domain, a central nucleotide-binding domain, and several C-terminal leucine-rich repeats. A significant effort in Dr. Vance’s lab focuses on the NAIP/NLRC4 inflammasomes, which are involved in the defense against bacterial pathogens.

To gain insight into the biology of inflammasome sensors, Dr. Vance and colleagues took advantage of the fact that mouse paralogs of neuronal apoptosis inhibitory protein (NAIP), an NLR family member, share a high degree of secondary structure homology and very similar domain architecture, yet they recognize distinct bacterial ligands.

As part of this work, investigators in Dr. Vance’s group generated 43 chimeric constructs between murine paralogs that recognize different ligands, and analyzed them for their ability to induce inflammasome assembly in response to FlaA, the Legionella pneumophila flagellin, or PrgJ, the Salmonella typhimurium type III secretion system inner rod protein.

“Our goal was to explore the molecular and biochemical mechanisms by which these sensors detect stimuli, and to understand how that detection event orchestrates the assembly of these complexes,” explains Dr. Vance. Monitoring inflammasome reconstitution by native gel electrophoresis revealed that ligand recognition requires several alpha helical domains from the nucleotide-binding domain, but does not require the leucine-rich repeat domain.

“Whereas everybody assumed that these sensors would use the leucine-rich repeats—and many of them probably do—in our case this was not required,” informs Dr. Vance. “Only the helical domains were used for this purpose.”

Inflammasome sensors are highly conserved across species. In plants, where inflammasomes have a similar domain combination, the leucine-rich repeats of the sensor molecule were shown to be important for recognition. “Plant inflammasomes have very different mechanisms of activation,” notes Dr. Vance. “In mammals, we are only beginning to appreciate sensor adaptability and the ability of sensors to detect pathogens in many different ways.”

A major gap in understanding the function of the inflammasomes stems from the insufficient knowledge about their biology, including their assembly, three-dimensional organization, and cellular dynamics. “Right now we can observe the end point, whether the inflammasome has assembled or not,” states Dr. Vance. “Reconstituting the complex with purified proteins in vitro to study its components and the various assembly steps will be a major focus for future work.”

Steroid Resistance in Leukemia

“Because patients whose leukemia cells are resistant to glucocorticoids have a lower cure rate, we are focused on finding out why some patients with acute lymphoblastic leukemia (ALL) are resistant to steroids, and our genomewide approach led us to the inflammmasome,” says William E. Evans, Pharm.D., endowed chair of pharmacogenomics at St. Jude Children’s Research Hospital. Dr. Evans and colleagues recently examined the molecular and cellular basis of glucocorticoid resistance in patients diagnosed with ALL, and found that leukemia cells from these patients had significantly higher expression of two proinflammatory genes, CASP1, which encodes caspase 1, and NLRP3.

Overexpression of CASP1 was associated with cleavage of the glucocorticoid receptor and increased glucocorticoid resistance. When CASP1 was knocked down with a short hairpin RNA or inhibited with the inhibitory protein CrmA, glucocorticoid receptor levels increased and glucocorticoid resistance decreased significantly (~40-fold).

A unique finding was that caspase 1 cleaves the glucocorticoid receptor, an action that had previously been unknown. “We showed that as soon as caspase 1 cleaves the receptor, cellular levels of glucocorticoid receptor decrease and cells become more resistant to all the effects of glucocorticoids,” details Dr. Evans. For example, cells resist glucocorticoid effects on gene transcription and the cytotoxic effects of glucocorticoids in leukemia cells.

This work also revealed that inflammasome activation and caspase 1 overexpression occur primarily in ALL cells with decreased methylation of CpG sites in both the CASP1 and NLRP3 gene promoters, as compared to steroid-sensitive leukemia cells. “We think that this mechanism explains about a third of the resistance to steroids that we see in leukemia patients,” continues Dr. Evans. “We hope to next find a caspase 1 small molecule inhibitor that can reverse this form of resistance when administered together with steroids.”

Previous clinical trials conducted with small molecule caspase 1 inhibitors did not have positive outcomes (for treatment of uncontrolled epilepsy), and a major research effort that Dr. Evans and colleagues are pursuing seeks to identify small molecule inhibitors of caspase 1. “We will first perform in vitro screens to test drugs that are already FDA-approved for other indications,” remarks Dr. Evans. “And we will then test them in cell lines and mouse xenograft models that we already generated.”

These findings not only stand to improve our understanding of drug resistance in leukemia and lymphoma, they also promise to impact other research fields. “We hope that our work will stimulate researchers working in other areas where glucocorticoids are prescribed,” concludes Dr. Evans. “We have already initiated interactions with investigators studying other diseases where inflammasome activation is known to occur.”

Chronic Disorders

“There is a tremendous amount of literature from numerous groups, showing that the inflammasome is not only activated, but is actually critical in driving the development of many chronic disorders, including Alzheimer’s disease, atherosclerosis, arthritis, and obesity,” says Jayakrishna Ambati, M.D., professor and vice chair of ophthalmology and visual sciences at the University of Kentucky. The literature also indicates, Dr. Ambati adds, that inhibiting the inflammasome “will be beneficial for reversing the disease state.”

Several years ago, Dr. Ambati and colleagues reported that in humans with age-related macular degeneration, retinal pigmented epithelial cells show an accumulation of Alu element transcripts, which are the most frequent small interspersed repetitive element found in the human genome. Accumulation of Alu RNA resulted from a defect in the microRNA-processing enzyme DICER1.

“This was the first time when Alu RNA was found to accumulate in patients with macular degeneration,” notes Dr. Ambati. Dr. Ambati’s group found that the NLRP3 inflammasome becomes activated in the cells with Alu RNA accumulation, and additional experiments implicated caspase 8 as a critical mediator of pathogenesis. “Subsequently, we found that antiretroviral drugs can block the inflammasome pathway in these cells,” Dr. Ambati points out.

Dr. Ambati and collaborators recently reported that a class of reverse transcriptase inhibitors is able to block inflammasome activation independently of their reverse transcription inhibition. “Currently, the biggest challenge is going ahead with clinical trials, because this is a chronic disease that takes years to develop, and identifying the best patient population, in a time frame that is feasible, is a concern when studying macular degeneration and chronic disorders in general,” states Dr. Ambati.

Mouse models of several inflammatory conditions, including geographic atrophy and graft-versus-host disease, revealed that these retroviral compounds prevented the activation of NLRP3 and caspase 1. “From a biological perspective, the greatest mystery is how inflammasomes sense the various danger signals,” maintains Dr. Ambati.

While Dr. Ambati’s group is focusing on the Alu RNA-mediated activation of the inflammasomes in macular degeneration, inflammasome activation is central to the pathogenesis of several other medical conditions. “Amyloid beta in Alzheimer’s disease, cholesterol plaques in atherosclerosis, and other very diverse sets of danger signals activate the NLRP3 inflammasome,” observes Dr. Ambati, “and there aren’t too many examples of molecules or molecular platforms in biology that recognize such a diverse set of molecular instigators.”

Inflammasomes, which can be activated through multiple signals including live bacteria, microbial toxins, xeno-compounds, pathogen-associated molecular patterns (PAMPs), and damage-associated molecular pattern molecules (DAMPs), have been implicated in several hereditary and acquired diseases. [Adipogen Life Sciences]

Assembly and Activation

“The cell biology of the inflammasome is an understudied area, partly because certain reagents to conduct this type of analysis have not been available,” says John D. MacMicking, Ph.D., associate professor of microbial pathogenesis at Yale University School of Medicine. “A major area we need to understand is how the inflammasome operates within the three-dimensional space of the cell.”

Work from Dr. MacMicking’s lab identified guanylate binding protein 5 (GBP5), which selectively stimulates the assembly of the NLRP3 inflammasomes in mammalian macrophages in response to pathogenic bacteria. Mice with homozygous GBP5 deletions showed pronounced defects in the cleavage of caspase 1 and proinflammatory mediator signaling and impaired Nlrp3-dependent inflammatory responses.

“An important question is whether the cofactors needed to bring about inflammasome assembly are hardwired and constitutively present, or whether they are under inducible regulation,” notes Dr. MacMicking. Dissecting the assembly and the regulation of the inflammasome could help advance the development of novel therapeutic strategies.

Although most efforts to date have focused on the canonical pathway of inflammasome activation, a noncanonical inflammasome activation pathway was described more recently. In mice, one of these noncanonical inflammasomes senses and responds to polysaccharides or lipopolysaccharides from the gram-negative bacterial cell wall and activates caspase 11.

Until recently, it was thought that these bacterial components could be recognized only when they were outside the cell, where they bind the Toll-like receptor 4 on the cell surface. “However, now we know that lipopolysaccharides are also released into the cell, for example, after engulfment of gram-negative bacteria by macrophages, where they are then detected in the cytosol,” remarks Dr. MacMicking.

In response to cytosolic lipopolysaccharides, the murine noncanonical caspase 11 pathway, corresponding to human caspases 4 and 5, induces pyroptosis, a critical step during endotoxic shock in a mouse model of disease.

“Lipopolysaccharide is a major component of gram-negative sepsis and a relatively common cause of fatality in critical care units, such as surgical and burn units,” notes Dr. MacMicking. “These findings could open up pathways that help manage this particular syndrome.”

Many therapeutic targets that are directed against extracellular lipopolysaccharides have not been working well. “Maybe we need to look at therapeutic approaches that are directed against these targets intracellularly, which could have clinical and therapeutic potential,” speculates Dr. MacMicking.

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