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.

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

Molecular Connections Between Sleep Deprivation and Inflammation

Anyone who has travelled across time zones knows how unpleasant it is when the regular rhythm of your biological clock is disrupted. Jetlag results when the body’s internal clock, or circadian rhythm is out of sync with external cues for “day and “night”, resulting in insomnia, extreme tiredness, difficulty concentrating and various other unpleasant symptoms.

On the bright side, jetlag is at least a temporary misery that is usually over after a few days of acclimation to the new time zone. Long-term disruption of the natural sleep/wake cycle, such as encountered by frequent long-distance travellers, shift workers, or people with physiological conditions that affect circadian rhythms, can be much more debilitating. Longer term health effects that have been associated with constant disruption of circadian rhythms include, insomnia, concentration problems, and increased susceptibility to diseases associated with chronic inflammation such as cancer, diabetes and cardiovascular disease.

Despite the fact that many of the genes and proteins involved in central control of circadian rhythms are known, the reason for the implied association between circadian clock components and immune function is not understood. Recently, a paper was published in the July issue of PNAS that identified a potential link between a circadian clock component and chronic inflammation. Continue reading

Curcumin Moderates the Health Effects of Diesel Exhaust

The majority of the ground transporation in the United States runs on diesel fuel. It powers most of the fleet of large vehicles that travel around Wisconsin and across country moving products and people. My family uses diesel fuel in all the large tractors that plant and harvest the 1,200+ acres of crops on the farm as well in the semitrailer that hauls the final product to market. Diesel also fuels a small portion of the passenger vehicles that people drive every day in the United States, including the car that is my main mode of transport. However, exposure to diesel exhaust can have negative effects on human health, and recent reports have found an increased risk of lung cancer. While these studies documented heavy exposure levels (miners underground with diesel generators), people still wonder about the effect of exposure when behind a truck spewing dark exhaust. A recent PLoS ONE article examined the effect of diesel exhaust particles on mouse cardiovascular systems and how treating with curcumin attenuated the negative effects. Continue reading

Of Lysosomes, Glucocorticoids and Inflammation

In a recent article published in Science Signaling, Yuangheng He and colleagues asked how the weak alkaline compound chloroquine (CQ) enhances the anti-inflammatory effects of synthetic glucocorticoids like dexamethasone, which are used to treat a host of inflammatory and autoimmune diseases. In the process they explored the intersection of lysosomal degradation pathways and glucocorticoid receptor signaling. For these investigations, they needed tools such as reporters and protein tags that allowed sensitive and accurate detection of events in real time in a variety of cells and systems.

The work is fascinating and removes the lysosome from its pigeon-hole description of garbage can (or recycling center) of the cell and places it in the center of cell signaling. The work also is fascinating because it takes a systems-view of a biological question: How is it that the drug chloroquine just happens to influence glucocorticoid signaling? To answer this question the authors employ an amazing array of techniques and technologies to ask questions in several systems under a many different conditions. The result is a work that explains a lot, but like all good science raises new questions for us to scratch our heads over.

Here I review this paper using “sketchnotes” with emphasis on the research techniques the researchers used. Continue reading