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<sup...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.45 &#956;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&#160;</td>
			<td>Bio-Rad</td>
			<td>4561043</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well plate&#160;</td>
			<td>Corning</td>
			<td>07-200-82</td>
			<td></td>
		</tr>
		<tr>
			<td>18 G needle&#160;</td>
			<td>BD Biosciences</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>25 G needle&#160;</td>
			<td>BD Biosciences</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tube&#160;</td>
			<td>Fisher Scientific</td>
			<td>50-809-218</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer&#160;</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-cm2 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&#8211;caspase-1 antibody</td>
			<td>AdipoGen</td>
			<td>AG-20B-0042-C100</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti&#8211;caspase-11 antibody</td>
			<td>Novus Biologicals</td>
			<td>NB120-10454</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti&#8211;caspase-3 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9662</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti&#8211;caspase-7 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9492</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti&#8211;caspase-8 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4927</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti&#8211;caspase-9 antibody</td>
			<td>Cell Signaling Technology</td>
			<td>9504</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti&#8211;cleaved caspase-3 antibody&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>9661</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti&#8211;cleaved caspase-7 antibody&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>9491</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti&#8211;cleaved caspase-8 antibody&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>8592</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse HRP-conjugated secondary antibody&#160;</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>315-035-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit HRP-conjugated secondary antibody&#160;</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>111-035-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rat HRP-conjugated secondary antibody&#160;</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>112-035-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti&#8211;&#946;-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&#160;</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)&#160;</td>
			<td>Cytiva</td>
			<td>29083461</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2&#160;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&#160;</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&#160;</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&#160;</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&#160;</td>
			<td>Sigma</td>
			<td>D6171</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti&#8211;caspase-1 antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB6215</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti&#8211;caspase-8 antibody</td>
			<td>Enzo</td>
			<td>ALX-804-242</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidified incubator&#160;</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])&#160;</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&#160;</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&#160;</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&#160;</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&#160;</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&#160;</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&#160;</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&#160;</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&#160;</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&#160;</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&#160;</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,<...

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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

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

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated fatty acids, which might play a critical role in regulating their immune functions. Numerous studies have shown that various fatty acids, saturated or unsaturated, may possess different impacts on cell growth and function. However, the approaches used for fatty acid preparation vary, which may lead to non-physiological results. Serum albumin, a natural carrier for fatty acids in mammalian peripheral blood, is recommended for forming a conjugate complex with the sodium salt of fatty acids to study fatty acid function in mammalian cells, thus minimizing the toxicity of fatty acid soap. Thus, a simple, relatively quick heating and sonicating method is developed and presented here for BSA-fatty acid conjugate formation. We describe a protocol using saturated fatty acids, especially stearic acids to induce severe cell death in mouse bone-marrow derived macrophages. We further demonstrate that saturated fatty acid-induced cell death is positively associated with accumulated cellular ceramide levels. This method can be extended for studies of the impact of fatty acid on other mammalian cells.

We illustrate a straight-forward method to derive murine primary macrophages from bone marrow cells and a simple method to prepare BSA-fatty acid conjugates. Then we demonstrate that saturated fatty acids can induce macrophage cell death, and such cell death is positively associated with cellular accumulation of ceramide levels.

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated ...

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.

Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

We quantify epidermal cell death in frogs with chytridiomycosis using two methods. First, we use terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ histology to determine differences between clinically infected and uninfected animals. Second, we conduct a time series analysis of apoptosis over infection using a caspase 3/7 protein analysis.

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is ...

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 ...

Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we investigated immunological effects of a novel lipid-DNA immunostimulant in nanoparticle form from different administration routes in the mouse: oral, intranasal, subcutaneous, footpad, intraperitoneal, and intravenous. These injections will directly influence the immune response, and harvesting lymphatic tissues and analysis of dendritic cell (DC) activation in the tissues are crucial parts of these evaluations. The extraction of mediastinal lymph nodes (mLNs) is important but quite complex because of the size and location of this organ. A stepwise procedure for harvesting the inguinal lymph node (iLN), mLN, and spleen and analyzing DC activation by flow cytometry is described.

Experimental procedures for the subsequent extraction of lymphatic tissues to test lymphoid dendritic cell activation are described after treatment of an immunostimulating nanomaterial.

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we ...