We thank members of the Kanneganti lab for their comments and suggestions, and we thank J. Gullett, PhD, for scientific editing support. Work in our lab is supported by National Institutes of Health (NIH) grants AI101935, AI124346, AI160179, AR056296, and CA253095 (to T.-D.K.) and by the American Lebanese Syrian Associated Charities (to T.-D.K.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

T.-D.K. is a consultant for Pfizer.

Monitoring caspase cleavage and activation provides one of the most comprehensive pictures of innate immune PCD activation as part of the innate immune response. The protocol described here demonstrates a strategy to monitor caspase activation in response to IAV, HSV1, and F. novicida infections and the sterile trigger LPS + ATP, but numerous other stimuli can induce PCD and could be used in this method, as has been shown in several publications44,48,49,50,51,52,53,54,56,57,58,59,60,61,62. This procedure can also be used to characterize the innate immune PCD in response to new triggers, where the mechanisms of PCD remain unknown. Using a combination of wild-type and genetically deficient cells and comparing the activation of different caspases between these populations can provide new insights into the sensors and regulators involved in PCD in response to a given stimulus.
Several technical points should be considered when performing this method. First, as this method involves infecting or otherwise stimulating cells to induce cell death, it is important to use sterile techniques for the isolation of the cells and for the subsequent stimulations. Contamination can significantly affect the results, leading to caspase activation even in unstimulated conditions. To test for this, it is best to always include an unstimulated control when collecting the samples and loading gels for the caspase blots. Additionally, when plating the cells, it is important to ensure that the cells are counted accurately and that clumping does not occur. If the cells are clumped, inconsistent numbers of cells will be deposited in the wells, and the MOI and the amount of protein per well will be affected. When performing the western blots, it is good practice to compare the banding pattern of the uncleaved, pro-form of the caspase, as well as to monitor the cleaved form to ensure that consistent amounts of protein were collected and loaded across the wells. However, when there is a high level of caspase activation in one sample, this can cause a distortion in the signal, leading to what appears to be a reduction in the pro-form in the other samples, as we show with caspase-8 in HSV1 and F. novicida infections in this example (Figure 2B,C). This likely occurs due to uneven antibody binding caused by the presence of too much protein in a particular spot on the membrane, or because of imaging artifacts where the substrate becomes oversaturated in a single sample. Adjusting the antibody dilutions or imaging settings can help overcome this issue. Additionally, when establishing an infection or stimulation for the first time, using a time course can be beneficial to identify when the activation of each caspase occurs. Without a time course, the data may be misleading, as the individual time point selected could be too early or too late, causing caspase activation to be missed and inappropriate conclusions to be made about its involvement. Similarly, testing different MOIs for infectious stimuli can also be beneficial to identify the specific conditions under which caspase(s) are activated.
The electrophoresis step also requires precision. When preparing to load the samples onto the gel, the tubes should always be centrifuged first, and only the supernatant should be loaded. Insoluble protein can interfere with the running of the gel and the subsequent analysis of the data. The combined supernatant and protein lysate should be used for the detection of caspase-1 rather than a pure cell lysate; this will greatly improve the sensitivity of detection. Caspase-3, caspase-7, and caspase-8 can also be detected well in the combined supernatant and protein lysate. However, for best results when detecting caspase-11 and caspase-9, the protein lysate collected in the RIPA buffer is sufficient. As the relative abundance of each caspase within the lysate can be low, as much sample as possible should be loaded onto the gel. This requires slow and steady pipetting to avoid overflowing into the neighboring wells. If poor separation is observed between the pro- and cleaved forms of the caspases, the gel run time can be extended, or a lower percentage of acrylamide can be used to optimize the separation.
Finally, the antibodies listed in this protocol have been optimized for use with mouse BMDMs, and several groups have rigorously tested them using genetically deficient cells as controls to confirm there are no non-specific bands at the molecular weights of interest. While other antibodies may also work, the ones listed here are optimal. Additionally, many of these antibodies also work well in tissue lysates, and other cell populations can also be collected and stimulated in vitro for analysis. Using cell lines or primary cells from humans or other species is also possible but will require an additional assessment of the antibodies for optimization. Human anti-caspase-1 (1:1,000) and human anti-caspase-8 (1:1,000; see Table of Materials) have been successfully used for such assessments, and the same anti-caspase-3, anti-cleaved caspase-3, anti-caspase-7, and anti-cleaved caspase-7 antibodies included in this protocol can be used for both mouse and human cells44,56,62.
The importance of understanding PCD pathways, including pyroptosis, apoptosis, necroptosis, and PANoptosis, as an integral component of the innate immune response is growing, as crosstalk between PCD pathways is increasingly being characterized, and cell death molecules are being identified as therapeutic targets across the disease spectrum3,4. Continuing to identify the innate immune sensors that induce PCD as well as III-PCD and characterize the timing and interplay of caspase activation will improve the ability to therapeutically modulate innate immune PCD pathways and improve patient outcomes.

The innate immune system acts as the first line of defense during infection and in response to sterile stimuli, such as tissue injury and alterations in homeostasis. Innate immune sensors on the cell surface and in the cytoplasm respond to pathogen- or damage-associated molecular patterns (PAMPs or DAMPs, respectively) to trigger inflammatory signaling pathways and cellular responses. One of the key processes of the innate immune response is the induction of cell death to remove infected or damaged cells and drive further innate and adaptive immune responses. Programmed cell death (PCD) is a highly conserved process across species, highlighting its evolutionary importance as an innate immune mechanism.
There are several innate immune PCD pathways that can be activated in all cell types. Caspases are a key family of highly conserved, intracellular, cysteine-dependent proteases that are critical across many PCD pathways, including the traditionally non-inflammatory apoptosis pathway, as well as inflammatory PCD pathways such as pyroptosis, necroptosis, and PANoptosis1,2,3,4,5. There are 11 human and 10 murine caspases that are well defined, as well as pseudo-caspases that may be functional, and most are constitutively expressed as inactive monomeric or dimeric pro-caspases that require cleavage for activation6,7. Caspases also contain important domains for the recruitment and formation of multiprotein complexes. These include the caspase activation and recruitment domain (CARD), which can be found in caspase-1, caspase-2, caspase-4, caspase-5, caspase-9, and caspase-11, or the death effector domain (DED), which is found in caspase-8 and caspase-10. Through both their proteolytic activity and their ability to form multiprotein complexes, caspases are critical drivers of innate immune PCD.
The role of caspases in innate immune PCD was first identified in apoptosis, where the initiator caspases, caspase-2, caspase-8, caspase-9, and caspase-10, activate the executioner caspases, caspase-3, caspase-6, and caspase-7, to drive cell death8,9,10,11,12. Initiator caspases can be activated by diverse signaling cascades; the extrinsic pathway activates caspase-8 through extracellular ligand-induced death receptor signaling, and the intrinsic pathway activates caspase-9 through the disruption of mitochondrial integrity13. Activated initiator caspases cleave the linker separating the large and small catalytic subunits of executioner caspases to produce their active forms. The executioner caspases then cleave their substrates to disassemble the cell, resulting in DNA degradation, membrane blebbing, nuclear fragmentation, and the release of apoptotic bodies14,15. This process typically ends in a non-lytic and non-inflammatory form of cell death when coupled with the immediate clearance of the dying cells by efferocytosis16. However, defects in efferocytosis or a lack of phagocytic cells can lead to the accumulation of apoptotic cells, which then undergo lytic and inflammatory cell death17,18.
The inflammatory caspases, including caspase-1 (human and mouse), caspase-4 and caspase-5 (human), and caspase-11 (mouse), have been discovered to be activated during a form of inflammatory innate immune PCD (III-PCD) called pyroptosis. Caspase-1 activation is associated with the formation of inflammasomes, which are multiprotein complexes containing a cytosolic innate immune sensor, an adaptor molecule (apoptosis-associated speck-like protein containing a CARD [ASC]), and caspase-1. The formation of this complex allows caspase-1 to undergo proximity-mediated autoproteolysis to release its active form, which can cleave target substrates including the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 and the pore-forming molecule gasdermin D (GSDMD)19,20,21,22,23. Caspase-11, caspase-4, and caspase-5 can also activate GSDMD without the upstream formation of the inflammasome after sensing PAMPs such as lipopolysaccharide (LPS)19,20. These caspases undergo dimerization followed by oligomerization and self-cleavage for activation upon binding to cytosolic LPS, which leads to non-canonical inflammasome activation24,25,26&#160;and caspase-1 activation in a cell-intrinsic manner to induce IL-1&#946; and IL-18 maturation20. The maturation and release of these pro-inflammatory cytokines characterize these caspases as &#34;inflammatory.&#34;&#160;Additionally, the apoptotic caspase-8 has been found to localize to the inflammasome, providing a link between apoptotic and pyroptotic processes.&#160;Studies have found that the apoptotic caspase-8 is also critical for regulating another form of PCD&#160;called necroptosis. The loss of caspase-8 results in spontaneous receptor-interacting serine-threonine kinase 3 (RIPK3)-mediated mixed lineage kinase domain-like pseudokinase (MLKL) activation to drive the III-PCD pathway of&#160;necroptosis27,28,29,30,31,32,33,34,35.&#160;
While caspases have historically been classified as &#34;apoptotic&#34; or &#34;inflammatory&#34; based on the type of cell death they initiate, growing evidence suggests there is extensive crosstalk between the innate immune PCD pathways through caspases3,4. For instance, the inflammatory caspase-1 from inflammasomes cleaves the apoptotic caspase-7 at its canonical activation site34. Caspase-1 activation can also lead to the cleavage of apoptotic substrates such as poly(ADP-ribose) polymerase 1 (PARP1)36. In cells lacking GSDMD, caspase-1 can also cleave caspase-337,38. Additionally, the canonically apoptotic caspase-3 can cleave gasdermin E (GSDME) to induce PCD17,18&#160;and also processes GSDMD into an inactive form40. Furthermore, caspase-8 recruitment to the inflammasome complex has been observed39,40,41,42,43,44,45, and caspase-8 is a key regulator of canonical and noncanonical inflammasome activation39. There are also overlapping and redundant roles for caspase-8 and caspase-1 in many inflammatory conditions, and innate immune PCD characterized by the activation of pyroptotic, apoptotic, and necroptotic components occurs across the disease spectrum39,46,47,48,49,50.
Based on this crosstalk between inflammatory and apoptotic caspases, a key gap in the mechanistic understanding of innate immunity and PCD was identified, leading to the discovery of PANoptosis. PANoptosis is a unique form of III-PCD that is activated in response to pathogens, PAMPs, DAMPs, and alterations in homeostasis and is regulated by PANoptosomes - multifaceted macromolecular complexes that integrate components from other cell death pathways44,50,51,52,53,54,55. The totality of the biological effects in PANoptosis cannot be individually accounted for by pyroptosis, apoptosis, or necroptosis alone3,4,35,36,39,46,47,48, as PANoptosis is characterized by the activation of multiple caspases, including caspase-1, caspase-11, caspase-8, caspase-9, caspase-3, and/or caspase-7, depending on the context44,48,49,50,51,52,53,54,56,57,58,59,60,61,62. PANoptosis has been increasingly implicated in infectious and inflammatory diseases, as well as in cancers and cancer therapies3,4,35,36,39,44,46,47,48,49,50,51,52,53,54,56 
,57,58,59,60,61,62,63,64,65,66.
Given the essential role of caspases across cell death pathways, including in apoptosis, pyroptosis,&#160;necroptosis, and PANoptosis, it is important to develop techniques to characterize their activation and understand the full complexity of the PCD pathways. The protocol here details a method to stimulate cells and measure the subsequent activation of caspases (Figure 1). This method leverages the proteolytic cleavage of caspases, which is generally required for their activation, as a means to study them. Through western blotting, the protein sizes can be determined, allowing for the clear visualization and differentiation of inactive pro-caspases and their activated, cleaved forms.
The major advantages of this protocol are 1) its ability to assess the activation of multiple caspases in parallel from a single population of endogenous cells to more accurately determine PCD activation and 2) the use of relatively simple lab techniques that do not require extensive training or expensive equipment. Previous protocols have used western blotting, fluorescent reporters, or antibody staining to monitor caspase activation in culture supernatants, cell and tissue lysates, whole cells via microscopy, and in vivo67,68,69,70,71, but these techniques generally only monitor one or two caspases in a sample. Furthermore, while synthetic peptide substrates containing caspase cleavage sites that fluoresce upon cleavage have been used to monitor caspase activation in cell or tissue lysates69, these substrates can often be cleaved by more than one caspase, making it difficult to determine the specific activation of individual caspases in this system. Additionally, the use of western blotting rather than the use of fluorescent reporters or other tag-based methods allows researchers to use endogenous cells rather than creating specific cell lines with reporter genes. There are multiple advantages to using endogenous cells, including the fact that many immortalized cell lines are deficient in key cell death molecules72,73, which could affect the results. Additionally, using endogenous cells allows for the evaluation of diverse cell types, such as macrophages, epithelial cells, and endothelial cells, rather than a single lineage. Western blotting is also a relatively simple and cost-effective technique that can be carried out in labs around the world without the need for large, expensive equipment or complicated setups.
This protocol is widely applicable across biology to understand both the cell death-dependent and cell death-independent functions of caspases, including their scaffolding roles and functions in other inflammatory signaling pathways74. Applying this method allows for a unified approach in the study of innate immune PCD pathways and inflammatory signaling across diseases and conditions, and this protocol can be used to identify critical regulatory processes and mechanistic connections that will inform the development of future therapeutic strategies.

<table><tbody><tr><td>0.45 &amp;mu;m filter</td><td>Millipore</td><td>SCHVU05RE</td><td></td></tr><tr><td>10 mL syringe</td><td>BD Biosciences</td><td>309604</td><td></td></tr><tr><td>12% polyacrylamide gel with 10 wells&amp;nbsp;</td><td>Bio-Rad</td><td>4561043</td><td></td></tr><tr><td>12-well plate&amp;nbsp;</td><td>Corning</td><td>07-200-82</td><td></td></tr><tr><td>18 G needle&amp;nbsp;</td><td>BD Biosciences</td><td>305195</td><td></td></tr><tr><td>25 G needle&amp;nbsp;</td><td>BD Biosciences</td><td>305122</td><td></td></tr><tr><td>50 mL tube&amp;nbsp;</td><td>Fisher Scientific</td><td>50-809-218</td><td></td></tr><tr><td>70 &amp;mu;m cell strainer&amp;nbsp;</td><td>Corning</td><td>431751</td><td></td></tr><tr><td>150 mm tissue culture dishes</td><td>Corning</td><td>430597</td><td></td></tr><tr><td>182-cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask</td><td>Genesee Scientific</td><td>25-211</td><td></td></tr><tr><td>Accessory white trans tray</td><td>Cytiva</td><td>29-0834-18</td><td></td></tr><tr><td>Anti&amp;ndash;caspase-1 antibody</td><td>AdipoGen</td><td>AG-20B-0042-C100</td><td></td></tr><tr><td>Anti&amp;ndash;caspase-11 antibody</td><td>Novus Biologicals</td><td>NB120-10454</td><td></td></tr><tr><td>Anti&amp;ndash;caspase-3 antibody</td><td>Cell Signaling Technology</td><td>9662</td><td></td></tr><tr><td>Anti&amp;ndash;caspase-7 antibody</td><td>Cell Signaling Technology</td><td>9492</td><td></td></tr><tr><td>Anti&amp;ndash;caspase-8 antibody</td><td>Cell Signaling Technology</td><td>4927</td><td></td></tr><tr><td>Anti&amp;ndash;caspase-9 antibody</td><td>Cell Signaling Technology</td><td>9504</td><td></td></tr><tr><td>Anti&amp;ndash;cleaved caspase-3 antibody&amp;nbsp;</td><td>Cell Signaling Technology</td><td>9661</td><td></td></tr><tr><td>Anti&amp;ndash;cleaved caspase-7 antibody&amp;nbsp;</td><td>Cell Signaling Technology</td><td>9491</td><td></td></tr><tr><td>Anti&amp;ndash;cleaved caspase-8 antibody&amp;nbsp;</td><td>Cell Signaling Technology</td><td>8592</td><td></td></tr><tr><td>Anti-mouse HRP-conjugated secondary antibody&amp;nbsp;</td><td>Jackson ImmunoResearch Laboratories</td><td>315-035-047</td><td></td></tr><tr><td>Anti-rabbit HRP-conjugated secondary antibody&amp;nbsp;</td><td>Jackson ImmunoResearch Laboratories</td><td>111-035-047</td><td></td></tr><tr><td>Anti-rat HRP-conjugated secondary antibody&amp;nbsp;</td><td>Jackson ImmunoResearch Laboratories</td><td>112-035-003</td><td></td></tr><tr><td>Anti&amp;ndash;&amp;beta;-Actin antibody (C4) HRP</td><td>Santa Cruz</td><td>sc-47778 HRP</td><td></td></tr><tr><td>ATP</td><td>InvivoGen</td><td>tlrl-atpl</td><td></td></tr><tr><td>BBL Trypticase Soy Broth</td><td>BD Biosciences</td><td>211768</td><td></td></tr><tr><td>Bead bath</td><td>Chemglass Life Sciences</td><td>CLS-4598-009</td><td></td></tr><tr><td>Biophotometer D30</td><td>Eppendorf</td><td>6133000010</td><td></td></tr><tr><td>BME</td><td>Sigma</td><td>M6250</td><td></td></tr><tr><td>Bromophenol blue&amp;nbsp;</td><td>Sigma</td><td>BO126</td><td></td></tr><tr><td>Cell scrapers</td><td>CellTreat Scientific Products</td><td>229315</td><td></td></tr><tr><td>Chemiluminescence imager (Amersham 600)&amp;nbsp;</td><td>Cytiva</td><td>29083461</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;chamber</td><td>VetEquip</td><td>901703</td><td></td></tr><tr><td>Cuvettes</td><td>Fisher Scientific</td><td>14-955-129</td><td></td></tr><tr><td>Dissecting scissors</td><td>Thermo Fisher Scientific</td><td>221S</td><td></td></tr><tr><td>DMEM</td><td>Thermo Fisher Scientific</td><td>11995-073</td><td></td></tr><tr><td>DTT</td><td>Sigma</td><td>43815</td><td></td></tr><tr><td>Eelectrophoresis apparatus&amp;nbsp;</td><td>Bio-Rad</td><td>1658004</td><td></td></tr><tr><td>Ethanol</td><td>Pharmco</td><td>111000200</td><td></td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Biowest</td><td>S1620</td><td></td></tr><tr><td>Filter paper</td><td>Bio-Rad</td><td>1703965</td><td></td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>22-327379</td><td></td></tr><tr><td>Francisella novicida (U112 strain)</td><td>BEI Resources</td><td>NR-13</td><td></td></tr><tr><td>Gel releaser&amp;nbsp;</td><td>Bio-Rad</td><td>1653320</td><td></td></tr><tr><td>Gentamycin</td><td>Gibco</td><td>15750060</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G7893</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G8898</td><td></td></tr><tr><td>HCl</td><td>Sigma</td><td>H9892</td><td></td></tr><tr><td>Heat block</td><td>Fisher Scientific</td><td>23-043-160</td><td></td></tr><tr><td>Herpes simplex virus 1 (HF strain)</td><td>ATCC</td><td>VR-260</td><td></td></tr><tr><td>High glucose DMEM&amp;nbsp;</td><td>Sigma</td><td>D6171</td><td></td></tr><tr><td>Human anti&amp;ndash;caspase-1 antibody</td><td>R&amp;amp;D Systems</td><td>MAB6215</td><td></td></tr><tr><td>Human anti&amp;ndash;caspase-8 antibody</td><td>Enzo</td><td>ALX-804-242</td><td></td></tr><tr><td>Humidified incubator&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>51026282</td><td></td></tr><tr><td>Image analysis software</td><td>ImageJ</td><td>v1.53a</td><td></td></tr><tr><td>IMDM</td><td>Thermo Fisher Scientific</td><td>12440-053</td><td></td></tr><tr><td>Influenza A virus (A/Puerto Rico/8/34, H1N1 [PR8])&amp;nbsp;</td><td>constructed per Hoffmann et al.</td><td></td><td></td></tr><tr><td>L929 cells</td><td>ATCC</td><td>CCL-1</td><td>cell line for creating L929-conditioned media</td></tr><tr><td>L-cysteine&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>BP376-100</td><td></td></tr><tr><td>Luminata Forte Western HRP substrate</td><td>Millipore</td><td>WBLUF0500</td><td>standard-sensitivity HRP substrate</td></tr><tr><td>MDCK cells</td><td>ATCC</td><td>CCL-34</td><td>cell line for determining IAV viral titer</td></tr><tr><td>Methanol</td><td>Sigma</td><td>322415</td><td></td></tr><tr><td>Microcentrifuge</td><td>Thermo Fisher Scientific</td><td>75002401</td><td></td></tr><tr><td>Non-essential amino acids&amp;nbsp;</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>Nonfat dried milk powder</td><td>Kroger</td><td></td><td></td></tr><tr><td>NP-40 solution&amp;nbsp;</td><td>Sigma</td><td>492016</td><td></td></tr><tr><td>PBS</td><td>Thermo Fisher Scientific</td><td>10010023</td><td></td></tr><tr><td>Penicillin and streptomycin&amp;nbsp;</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>Petri dish</td><td>Fisher Scientific</td><td>07-202-011</td><td></td></tr><tr><td>PhosSTOP</td><td>Roche</td><td>PHOSS-RO</td><td></td></tr><tr><td>Power source&amp;nbsp;</td><td>Bio-Rad</td><td>164-5052</td><td></td></tr><tr><td>Protease inhibitor tablet</td><td>Sigma</td><td>S8820</td><td></td></tr><tr><td>PVDF membrane&amp;nbsp;</td><td>Millipore</td><td>IPVH00010</td><td></td></tr><tr><td>Rocking shaker</td><td>Labnet</td><td>S2035-E</td><td></td></tr><tr><td>SDS</td><td>Sigma</td><td>L3771</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma</td><td>30970</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma</td><td>72068</td><td></td></tr><tr><td>Sodium pyruvate&amp;nbsp;</td><td>Gibco</td><td>11360-070</td><td></td></tr><tr><td>Square Petri dish</td><td>Fisher Scientific</td><td>FB0875711A</td><td></td></tr><tr><td>Stripping buffer</td><td>Thermo Fisher Scientific</td><td>21059</td><td></td></tr><tr><td>Super Signal Femto HRP substrate</td><td>Thermo Fisher Scientific</td><td>34580</td><td>high-sensitivity HRP substrate</td></tr><tr><td>Tabletop centrifuge</td><td>Thermo Fisher Scientific</td><td>75004524</td><td></td></tr><tr><td>Trans-Blot semi-dry system&amp;nbsp;</td><td>Bio-Rad</td><td>170-3940</td><td></td></tr><tr><td>Tris</td><td>Sigma</td><td>TRIS-RO</td><td></td></tr><tr><td>Tween 20&amp;nbsp;</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>Ultrapure lipopolysaccharide (LPS) from E. coli 0111:B4</td><td>InvivoGen</td><td>tlrl-3pelps</td><td></td></tr><tr><td>Vero cells</td><td>ATCC</td><td>CCL-81</td><td>cell line for determining HSV1 viral titer</td></tr></tbody></table>

evaluation of caspase activation to assess innate immune cell death

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is the initiation of innate immune programmed cell death (PCD) to eliminate infected or damaged cells and propagate immune responses. However, excess PCD is associated with inflammation and pathology. Therefore, understanding the activation and regulation of PCD is a central aspect of characterizing innate immune responses and identifying new therapeutic targets across the disease spectrum.
This protocol provides methods for characterizing innate immune PCD activation by monitoring caspases, a family of cysteine-dependent proteases that are often associated with diverse PCD pathways, including&#160;apoptosis,&#160;pyroptosis, necroptosis, and PANoptosis. Initial reports characterized caspase-2, caspase-8, caspase-9, and caspase-10 as initiator caspases and caspase-3, caspase-6, and caspase-7 as effector caspases in apoptosis, while later studies found the inflammatory caspases, caspase-1, caspase-4, caspase-5, and caspase-11, drive pyroptosis. It is now known that there is extensive crosstalk between the caspases and other innate immune and&#160;cell death molecules across the previously defined PCD pathways, identifying a key knowledge gap in the mechanistic understanding of innate immunity and PCD and&#160;leading to the characterization of PANoptosis. PANoptosis is a unique innate immune inflammatory PCD pathway regulated by PANoptosome complexes, which integrate components, including caspases,&#160;from other cell death pathways.
Here, methods for assessing the activation of caspases in response to various stimuli are provided. These methods allow for the characterization of PCD pathways both in vitro and in vivo, as activated caspases undergo proteolytic cleavage that can be visualized by western blotting using optimal antibodies and blotting conditions. A protocol and western blotting workflow have been established that allow for the assessment of the activation of multiple caspases from the same cellular population, providing a comprehensive characterization of the PCD processes. This method can be applied across research areas in development, homeostasis, infection, inflammation, and cancer to evaluate PCD pathways throughout cellular processes in health and disease.

PANoptosis has been observed in response to numerous bacterial, viral, and fungal infections and other inflammatory stimuli, as well as in cancer cells44,48,49,50,51,52,53,54,56,57,58,60,61,62. In these cases, the activation of multiple caspases has been reported. Using the example stimulations in this protocol that are known to induce PANoptosis, namely IAV, HSV1, and F. novicida44,48,50,51,53,57, it is expected that cleavage may be observed as a surrogate for the activation of multiple caspases (Figure 2A-C). Additionally, LPS + ATP is included as a control; this combination is a canonical NLRP3 inflammasome trigger that is expected to induce caspase-1 activation. The activation of caspase-1 can be visualized in response to each of these triggers by the cleavage of the p45 pro-form to the p20 active form (Figure 2D). The p20 form can then cleave GSDMD and initiate cell death19.
Similarly, the activation of the apoptotic initiator caspase, caspase-8, is observed, as denoted by the presence of the p18 cleaved form (Figure 2A-D). The weak activation of caspase-9 can also be observed in some cases with these stimuli (Figure 2A-D). Downstream of these initiators, the effector caspases caspase-3 and caspase-7 are activated; this activation is observed as the formation of the p17/19 cleaved form of caspase-3 and the p20 cleaved form of caspase-7 (Figure 2A-D). To date, the activation of caspase-11 has not been widely evaluated in PANoptosis, but its activation has been observed in some contexts54. Caspase-11 remains an important inflammatory caspase, and its activation can be observed by the formation of its p26 cleaved form. While some low-level caspase-11 activation is seen, robust activation is not observed in response to IAV, HSV1, or F. novicida infection or LPS + ATP stimulation (Figure 2A-D).
Cells lacking upstream PANoptosis sensors do not undergo cell death in response to PANoptosis-inducing stimuli. For example, IAV infection induces the formation of the ZBP1-PANoptosome48,51,53,57, and Zbp1-/- cells are protected from cell death during IAV infection (Figure 3A). Similarly, HSV1 and F. novicida infections induce the formation of the AIM2-PANoptosome44, and&#160;Aim2-/- cells fail to undergo robust cell death in response to these infections (Figure 3B,C). In cells that are deficient in the upstream sensor necessary for PANoptosome formation, the activation of caspases is significantly reduced or even eliminated, as can be seen by the reduction in the intensity of the cleaved bands in the western blots (Figure 2A-C). Similarly, cells lacking upstream inflammasome sensors are protected from cell death in response to their respective stimuli, and Nlrp3-/- cells do not undergo cell death in response to LPS + ATP (Figure 3D). These cells also show reduced caspase activation (Figure 2D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64308/64308fig01.jpg" /> 
Figure 1: Overview of the experimental procedure. Bone marrow is isolated and differentiated into bone marrow-derived macrophages. These cells are then stimulated with a trigger that activates innate immune signaling and cell death, such as infection or an inflammatory stimulus. After the cells activate and begin to undergo PCD, the lysate is collected and analyzed by western blotting to monitor caspase cleavage to their active forms. Abbreviations: BMDMs = bone marrow-derived macrophages; DAMPs = damage-associated molecular patterns; III-PCD = inflammatory innate immune programmed cell death;&#160;PAMPs = pathogen-associated molecular patterns; PCD = programmed cell death. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64308/64308fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64308/64308fig02.jpg" /> 
Figure 2: Activation of caspases in response to stimuli. Caspase activation from cell culture lysates can be assessed by evaluating the size of the products run on an electrophoretic gel. (A-D) Immunoblot analysis of pro- (P45) and activated (P20) CASP1, pro- (P43) and cleaved (P36 and P26) CASP11, pro- (P35) and cleaved (P17/P19) CASP3, pro- (P35) and cleaved (P20) CASP7, pro- (P55) and cleaved (P44 and P18) CASP8, and pro- (P49) and cleaved (P37) CASP9 in WT and Zbp1&#8722;/&#8722;, WT and Aim2&#8722;/&#8722;, or WT and Nlrp3&#8722;/&#8722; BMDMs after (A) IAV infection, (B) HSV1 infection, (C) Francisella novicida infection, or (D) LPS + ATP stimulation. Images are representative of three independent experiments. Abbreviations: BMDMs = bone marrow-derived macrophages; CASP1 = caspase-1; CASP3 = caspase-3; CASP7 = caspase-7; CASP8 = caspase-8; CASP9 = caspase-9; CASP11 = caspase-11; HSV1 = herpes simplex virus 1; IAV = influenza A virus; LPS = lipopolysaccharide; WT = wild-type. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64308/64308fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64308/64308fig03.jpg" /> 
Figure 3: Inhibition of cell death when caspase activation does not occur. (A-D) Representative images of cell death in WT and Zbp1&#8722;/&#8722;, WT and Aim2&#8722;/&#8722;, or WT and Nlrp3&#8722;/&#8722; BMDMs after (A) IAV infection, (B) HSV1 infection, (C) Francisella novicida infection, or (D) LPS + ATP stimulation. The red mask denotes dead cells. Scale bars = 50 &#181;m. Images are representative of three independent experiments. Abbreviations: BMDMs = bone marrow-derived macrophages; HSV1 = herpes simplex virus 1; IAV = influenza A virus; LPS = lipopolysaccharide; WT = wild-type. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64308/64308fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64308/64308_Table 1v2.xlsx" target="_blank">Please click here to download this table.</a>

Watch this Scientific Journal Video about Evaluation of Caspase Activation to Assess Innate Immune Cell Death at JoVE.com

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

This protocol describes a comprehensive method for assessing caspase activation (caspase-1, caspase-3, caspase-7, caspase-8, caspase-9, and caspase-11) in response to both in vitro and in vivo (in mice) models of infection, sterile insults, and cancer to determine the initiation of cell death pathways, such as pyroptosis, apoptosis, necroptosis, and PANoptosis.

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is ...

evaluation-of-caspase-activation-to-assess-innate-immune-cell-death

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

3.0K Views.  St. Jude Children's Research Hospital. This protocol describes a comprehensive method for assessing caspase activation (caspase-1, caspase-3, caspase-7, caspase-8, caspase-9, and caspase-11) in response to both in vitro and in vivo (in mice) models of infection, sterile insults, and cancer to determine the initiation of cell death pathways, such as pyroptosis, apoptosis, necroptosis, and PANoptosis.

Video: Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first to be activated in these pathways. This group includes caspase-2, -8, and -9, as well as the inflammatory caspases, caspase-1, -4, and -5. The initiator caspases are all activated by dimerization following recruitment to specific multiprotein complexes called activation platforms. Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based approach where split fluorescent proteins fused to initiator caspases are used to visualize the recruitment of initiator caspases to their activation platforms and the resulting induced proximity. This fluorescence provides a readout of one of the earliest steps required for initiator caspase activation. Using a number of different microscopy-based approaches, this technique can provide quantitative data on the efficiency of caspase activation on a population level as well as the kinetics of caspase activation and the size and number of caspase activating complexes on a per cell basis.

This protocol describes caspase Bimolecular Fluorescence Complementation (BiFC); an imaging-based method that can be used to visualize induced proximity of initiator caspases, which is the first step in their activation.

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first ...

Biochemistry

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1&#946;-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. 
Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

Here, an experimental workflow is presented that enables the detection of caspase-8 processing directly at the death-inducing signaling complex (DISC) and determines the composition of this complex. This methodology has broad applications, from unraveling the molecular mechanisms of cell death pathways to the dynamic modeling of apoptosis networks.

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ...

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially described to occur by apoptosis, programmed cell death is now known to encompass three interconnected pathways: pyroptosis, apoptosis, and necroptosis, together coined as one process, PANoptosis. Influence A virus (IAV) infection induces PANoptosis in mammalian cells by inducing the activation of different caspases, which, in turn, cleave various host as well as viral proteins, leading to processes like the activation of the host innate antiviral response or the degradation of antagonistic host proteins. In this regard, caspase 3-mediated cleavage of host cortactin, histone deacetylase 4 (HDAC4), and histone deacetylase 6 (HDAC6) has been discovered in both animal and human epithelial cells in response to the IAV infection. To demonstrate this, inhibitors, RNA interference, and site-directed mutagenesis were employed, and, subsequently, the cleavage or resistance to cleavage and the recovery of cortactin, HDAC4, and HDAC6 polypeptides were measured by western blotting. These methods, in conjunction with RT-qPCR, form a simple yet effective strategy to identify the host as well as viral proteins undergoing caspase-mediated cleavage during an infection of IAV or other human and animal viruses. The present protocol elaborates the representative results of this strategy, and the ways to make it more effective are also discussed.

Influenza A virus (IAV) infection activates the caspases that cleave host and viral proteins, which, in turn, have pro- and antiviral functions. By employing inhibitors, RNA interference, site-directed mutagenesis, and western blotting and RT-qPCR techniques, caspases in infected mammalian cells that cleave host cortactin and histone deacetylases were identified.

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially ...

Visualization of Caspase Activation in Human Monocyte-Derived Macrophages

Source: Bol&#237;var, B. E., et. al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63162">Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages.</a> J. Vis. Exp. (2022).
This video demonstrates the activation of caspase proteins in human macrophages using bimolecular fluorescence complementation reporter proteins. The pro-domains of caspase-1 are fused with non-fluorescent fragments of Venus proteins, which are recruited to inflammasome complexes. The proximity of inflammasomes induces the refolding and fluorescence of Venus fragments, confirming caspase activation in the macrophages.

Wizualizacja aktywacji kaspazy w ludzkich makrofagach pochodzących z monocytów

Source: Bol&#237;var, B. E., et. al., Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages. J. Vis. Exp. 
...

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Immune Modulation

Assessment of the Activation of Multiple Caspases in Macrophages

Source: Han, J. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64308">Evaluation of Caspase Activation to Assess Innate Immune Cell Death.</a>&#160;J. Vis. Exp.&#160;(2023)
This video demonstrates the assessment of multiple caspase activations in bone marrow-derived macrophages through stimulation with the influenza virus. Following macrophage lysis, heat-inactivation of lysate, and centrifugation, intracellular caspases are collected for subsequent immunological analysis.

Ocena aktywacji wielu kaspaz w makrofagach

1. Preparing the solutions

	Prepare L929-conditioned media.
	
		Plate 1 &#215; 106&#160;L929 cells (see&#160;Table of Materials) in an 182 ...

Immunodiagnostics

Cellular Immunodiagnostics

Caspases

Caspase, a family of cysteine proteases, serve as effectors in apoptosis. The ced3 gene in C.elegans was first identified to be involved in apoptosis. This gene encodes the ced-3 caspase that is similar to the interleukin-1-beta converting enzyme or ICE in mammals. In addition to apoptosis, caspases also function in the inflammatory response. Inflammatory caspases are essential in activating pro-inflammatory cytokines that recruit immune cells and block the replication of pathogens inside cells.
Caspase Activation
Caspases are produced as inactive zymogens, called procaspases. The procaspases contain four loops, L1 to L4. During activation, L2 is cleaved, and the monomeric subunits dimerize. The initiator procaspases also have long pro-domains such as the death effector domain (DED) or caspase recruitment domain (CARD). These pro-domains interact with the adaptor molecules to cleave and activate the initiator caspases. Conversely, executioner caspases have short pro-domains and are cleaved by the active initiator caspases.
Caspases and Homeostasis
Cell proliferation and cell death are both essential to maintain homeostasis in multicellular organisms. The over- and under-activation of caspases, thus, can lead to many diseases. For example, decreased caspase activity is a common feature of cancerous cells. Many infectious diseases also decrease caspase activity. For example, the p35 protein released by certain viruses binds the caspases, thus preventing caspase activation and blocking apoptosis. In contrast, the over-activation of inflammatory caspases causes sepsis due to increased inflammatory response. Excessive activation of caspases can also cause diseases such as Alzheimer&#39;s, Parkinson&#8217;s, and Huntington&#39;s.

Kaspazy

Caspase, a family of cysteine proteases, serve as effectors in apoptosis. The ced3 gene in C.elegans was first identified to be involved in apoptosis. ...