Activating the Inflammasome: A New Tool Brings New Understanding

Innate immunity, the first line of immune defense, uses a system of host pattern recognition receptors (PRRs) to recognize signals of “danger” including invariant pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). These signals in turn recruit and assemble protein complexes called inflammasomes, resulting in the activation of caspase-1, the processing and release of the pro-inflammatory cytokines IL-1ß and IL-18, and the induction of programmed, lytic cell death known as pyroptosis.

Innate immunity and the activity of the inflammasome are critical for successful immunity against a myriad of environmental pathogens. However dysregulation of inflammasome activity is associated with many inflammatory diseases including type 2 diabetes, obesity-induced asthma, and insulin resistance. Recently, aberrant NLRP3 inflammasome activity also has been associated with age-related macular degeneration and Alzheimer disease. Understanding the players and regulators involved in inflammasome activity and regulation may provide additional therapeutic targets for these diseases.

Currently inflammasome activation is monitored using antibody-based techniques such as Western blotting or ELISA’s to detect processed caspase-1 or processed IL-1ß. These techniques are tedious and are only indirect measures of caspase activity. Further, gaining information about kinetics—relating inflammasome assembly, caspase-1 activation and pyroptosis in time—is very difficult using these methods. O’Brien et al. describe a one-step, high-throughput method that enables the direct measurement of caspase-1 activity. The assay can be multiplexed with a fluorescent viability assay, providing information about the timing of cell death and caspase-1 activity from the same sample. Continue reading

Inflammasomes and Pyroptosis

In today’s post, guest blogger,  Martha O’Brien, PhD, provides a preview of her upcoming AAI poster and block symposium talk on the inflammasome, caspase-1 activity and pyroptosis.

Schematic of the Caspase-Glo 1 Inflammasome Assay.

Schematic of the Caspase-Glo 1 Inflammasome Assay.

Responding rapidly to microbial pathogens and damage-associated molecular markers is critical to our innate immune system. Caspase-1 is pivotal in this process leading to processing and release of essential cytokines and an immunogenic form of cell death, termed pyroptosis. Upon sensing pathogen-associated and damage-associated molecular patterns (PAMPs and DAMPs), innate immune cells form inflammasome protein complexes that recruit and activate caspase-1 (canonical inflammasomes). In addition, other inflammatory caspases, 4 and 5 in humans and 11 in mice, directly bind bacterial lipopolysaccharides (LPS), triggering pyroptosis (non-canonical inflammasome). LPS-triggered non-canonical inflammasomes in mice and humans ultimately lead to canonical inflammasome engagement and caspase-1 activation (1–3).  Caspase-1 was originally termed interleukin converting enzyme (ICE) for its well-established role in processing IL-1ß and IL-18, two important inflammation cytokines. How caspase-1 mediates pyroptosis is less well understood, but is beginning to be delineated. Recently, a substrate of the inflammatory caspases, gasdermin D, was identified and its processed fragment, gasdermin-N domain, was shown to be required for pyroptosis in non-canonical inflammasome circumstances (4, 5). The precise role of gasdermin D in canonical inflammasome-triggered pyroptosis is still under investigation. Linking inflammatory caspases directly to pyroptosis is a notable step in understanding the mechanism of this important form of cell death.

Pyroptosis is clearly one means of releasing processed IL-1ß and IL-18 from the cell. However depending on the cell type and stimulus, there is evidence for inflammasome engagement, caspase-1 activation, and release of IL-1ß in the absence of cell death (6, 7). On the flip-side there is also evidence for caspase-1 mediated pyroptosis that helps clear bacteria, independent of IL-1ß and IL-18 involvement (8). To enable further studies on the inflammasome and in particular, assessing the connections between caspase-1 activation, pyroptosis, and cytokine release, Promega developed a new tool to conveniently monitor caspase-1 activation, the Caspase-Glo® 1 Inflammasome Assay.  This bioluminescent, plate-based assay is used to measure caspase-1 activity directly in cell cultures or to monitor released caspase-1 activity in culture medium from treated cells. This flexibility allows easy multiplexing to monitor all three outcomes of inflammasome stimulation; caspase-1 activity, pyroptosis, and release of IL-1ß and IL-18. Caspase-1 activation typically is monitored indirectly with western blots of processed caspase-1. Now the activity of the enzyme can be monitored directly, providing accurate information on temporal aspects of the inflammasome. The assay can be readily combined with real-time measures of cell death (e.g., CellTox™ Green Cytotoxicity Assay) and some of the culture medium can be removed for IL-1ß/IL-18 assessment, leaving the cells and remaining culture medium for caspase-1 activity measurements. At the upcoming meeting of the American Association of Immunologists (AAI) in Seattle, May 13th-17th, oral and poster presentations will highlight use of the Caspase-Glo® 1 Inflammasome Assay and its value for exploring the relationship between inflammasomes and pyroptosis.

References

  1. Schmid-Burgk et al. (2015) Caspase-4 mediates non-canonical activation of the NLRP3 inflammasome in human myeloid cells. J. Immunol. 45, 2911–7.
  2. Baker et al. (2015) NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5. J. Immunol. 45, 2918–26.
  3. Ruhl, S. and P. Broz (2015) Caspase-11 activates a canonical NLRP3 inflammasome by promoting K+ Eur. J. Immunol. 45, 2927–36.
  4. Shi et al. (2015) Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–5.
  5. Kayagaki et al. (2015) Caspase-11 cleaves gasdermin D for non-canonical inflammasome signaling. Nature 526, 666–71.
  6. Gaidt et al. (2016) Human monocytes engage an alternative inflammasome pathway. Immunity 44, 833–46.
  7. Chen et al. (2014) The neutrophil NLRC4 inflammasome selectively promotes IL-1ß maturation without pyroptosis during acuteSalmonella Cell Reports 8, 570–82.
  8. Miao et al. (2010) Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria. Nature Immunology 11, 1136–42.

Inflammasome Research: A Tool to Aid Progress

Inflammasomes: A Few Basics

Inflammasomes are protein complexes composed of immune system receptors and sensor molecules. These complexes can respond to both infectious organisms and molecules derived from host proteins. When activated, a series of receptors and molecules signal via either pathogen-associate molecular patterns (PAMPs) induced by microbial pathogens, or danger-associated molecular patterns (DAMPs) induced as a result of endogenous stressors; the common next step in signaling is through pattern recognition receptors (PRR).

Inflammasome diagram.

Innate immune response in inflammation; a basic diagram.

Inflammasome activation is integral to the host immune response in mice and humans (1). The activation results in signaling that activates the caspase-1 scaffold, causing release of immune mediators such as interleukins IL-1β and IL-18. So, whether inflammation is host tissue- or pathogen-induced, inflammasome activation results in a cascade of receptor signaling and mediator release, of which caspase-1 is a critical component.

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Inflammasomes: Peeking Inside the Inflammatory Process

Most of us have experienced an inflammatory response at some point in our lives. Fever, achy joints, swelling around a scrape or cut, all of these are forms of inflammatory response. Inflammation is the body’s response to infection or tissue damage and acts to limit harm to the rest of the body. A key player in the inflammation process is a group of protein complexes call inflammasomes. The term “inflammasome” was first used in 2002 by researchers in Switzerland (1) to refer to a caspase-activating protein complex. We now know that inflammasomes are cytosolic multiprotein platforms that assemble in response to pathogens and other signals. Inflammasome assembly results in the processing of the inactive procaspase-1 into the active cysteine-protease enzyme, caspase-1. Caspase-1, in turn, activates the proinflammatory cytokines Interleukins IL-1β and IL-18. In addition, caspase-1 is also required for pyroptosis, which is an inflammatory form of cell death that combines the characteristics of apoptosis (fragmented DNA) and necrosis (inflammation and cytokine release) and is frequently associated with microbial infections.

Inflammasome complexes are made up of scaffolding sensor proteins (NLR, AIM2, ALR), and an adaptor protein containing a caspase activation and retention domain (CARD) and inactive procaspase-1. Most inflammasomes are formed with one or two NLRs (NOD-like receptors). However, non-NLR proteins such as AIM2 (absent in melanoma 2) and pyrin can also form inflammasomes. The different sensor proteins are activated by different types of outside stimuli, and inflammasomes are loosely sorted into families based on the protein forming these sensors.26061534-Inflamasome-Assay-Blog-figure-WEB Continue reading