Name: visualization of inflammatory caspases induced proximity in human monocyte-derived macrophages
Uploaded: 2022-04-06T12:45:00
Description: Watch this Scientific Journal Video about Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages at JoVE.com

We thank the members of LBH&#39;s lab past and present who contributed to the development of this technique. This lab is supported by NIH/NIDDK T32DK060445 (BEB), NIH/NIDDK F32DK121479 (BEB), NIH/NIGMS R01GM121389 (LBH). Figure 2 was drawn using Biorender software.

This protocol follows the guidelines of Baylor College of Medicine&#39;s human research ethics committee for the manipulation of human samples. Blood samples are handled following the institutional safety guidelines for human samples. Blood samples are obtained at a regional blood bank, where they are collected with citrate phosphate dextrose (CPD) solution. However, blood collected with other anticoagulants like sodium heparin, lithium heparin, or EDTA can also be used for this protocol28,</...

<ol>
	<li>Boatright, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unified+model+for+apical+caspase+activation.">A unified model for apical caspase activation.</a> Molecular Cell. 11 (2), 529-541 (2003).</li><li>Pop, C., Salvesen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+caspases:+activation,+specificity,+and+regulation.">Human caspases: activation, specificity, and regulation.</a> Journal of Biological Chemistry. 284 (33), 21777-21781 (2009).</li><li>Boice, A., Bouchier-Hayes, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+apoptotic+caspases+in+cancer.">Targeting apoptotic caspases in cancer.</a> Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1867 (6), 118688 (2020).</li><li>Bol&#237;var, B. E., Vogel, T. P., Bouchier-Hayes, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+caspase+regulation:+maintaining+balance+between+inflammation+and+cell+death+in+health+and+disease.">Inflammatory caspase regulation: maintaining balance between inflammation and cell death in health and disease.</a> The FEBS Journal. 286 (14), 2628-2644 (2019).</li><li>Martinon, F., Tschopp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+caspases:+linking+an+intracellular+innate+immune+system+to+autoinflammatory+diseases.">Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases.</a> Cell. 117 (5), 561-574 (2004).</li><li>Lamkanfi, M., Vishva, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functions+of+inflammasomes.">Mechanisms and functions of inflammasomes.</a> Cell. 157 (5), 1013-1022 (2014).</li><li>Vigan&#242;, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+caspase-4+and+caspase-5+regulate+the+one-step+noncanonical+inflammasome+activation+in+monocytes.">Human caspase-4 and caspase-5 regulate the one-step noncanonical inflammasome activation in monocytes.</a> Nature Communications. 6, 8761 (2015).</li><li>Cerretti, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+the+interleukin-1+beta+converting+enzyme.">Molecular cloning of the interleukin-1 beta converting enzyme.</a> Science. 256 (5053), 97 (1992).</li><li>van de Veerdonk, F. L., Netea, M. G., Dinarello, C. A., Joosten, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammasome+activation+and+IL-1&#946;+and+IL-18+processing+during+infection.">Inflammasome activation and IL-1&#946; and IL-18 processing during infection.</a> Trends in Immunology. 32 (3), 110-116 (2011).</li><li>Martinon, F., Burns, K., Tschopp, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inflammasome:+a+molecular+platform+triggering+activation+of+inflammatory+caspases+and+processing+of+proIL-beta.">The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta.</a> Molecular Cell. 10 (2), 417-426 (2002).</li><li>Kayagaki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncanonical+inflammasome+activation+targets+caspase-11.">Noncanonical inflammasome activation targets caspase-11.</a> Nature. 479 (7371), 117-121 (2011).</li><li>Kayagaki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncanonical+inflammasome+activation+by+intracellular+LPS+independent+of+TLR4.">Noncanonical inflammasome activation by intracellular LPS independent of TLR4.</a> Science. 341 (6151), 1246-1249 (2013).</li><li>Masumoto, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ASC,+a+novel+22-kDa+protein,+aggregates+during+apoptosis+of+human+promyelocytic+leukemia+HL-60+cells.">ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells.</a> The Journal of Biological Chemistry. 274 (48), 33835-33838 (1999).</li><li>Bertin, J., DiStefano, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PYRIN+domain:+a+novel+motif+found+in+apoptosis+and+inflammation+proteins.">The PYRIN domain: a novel motif found in apoptosis and inflammation proteins.</a> Cell Death and Differentiation. 7 (12), 1273-1274 (2000).</li><li>Agostini, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NALP3+forms+an+IL-1beta-processing+inflammasome+with+increased+activity+in+Muckle-Wells+autoinflammatory+disorder.">NALP3 forms an IL-1beta-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder.</a> Immunity. 20 (3), 319-325 (2004).</li><li>B&#252;rckst&#252;mmer, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+orthogonal+proteomic-genomic+screen+identifies+AIM2+as+a+cytoplasmic+DNA+sensor+for+the+inflammasome.">An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome.</a> Nature Immunology. 10 (3), 266-272 (2009).</li><li>Latz, E., Xiao, T. S., Stutz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+and+regulation+of+the+inflammasomes.">Activation and regulation of the inflammasomes.</a> Nature Reviews Immunology. 13 (6), 397-411 (2013).</li><li>Kayagaki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-11+cleaves+gasdermin+D+for+noncanonical+inflammasome+signalling.">Caspase-11 cleaves gasdermin D for noncanonical inflammasome signalling.</a> Nature. 526 (7575), 666-671 (2015).</li><li>Shi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+GSDMD+by+inflammatory+caspases+determines+pyroptotic+cell+death.">Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.</a> Nature. 526 (7575), 660-665 (2015).</li><li>Shi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+caspases+are+innate+immune+receptors+for+intracellular+LPS.">Inflammatory caspases are innate immune receptors for intracellular LPS.</a> Nature. 514 (7521), 187-192 (2014).</li><li>Bol&#237;var, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noncanonical+Roles+of+Caspase-4+and+Caspase-5+in+Heme-Driven+IL-1&#946;+Release+and+Cell+Death.">Noncanonical Roles of Caspase-4 and Caspase-5 in Heme-Driven IL-1&#946; Release and Cell Death.</a> The Journal of Immunology. 206 (8), 1878-1889 (2021).</li><li>Schmid-Burgk, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-4+mediates+noncanonical+activation+of+the+NLRP3+inflammasome+in+human+myeloid+cells.">Caspase-4 mediates noncanonical activation of the NLRP3 inflammasome in human myeloid cells.</a> European Journal of Immunology. 45 (10), 2911-2917 (2015).</li><li>Baker, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NLRP3+inflammasome+activation+downstream+of+cytoplasmic+LPS+recognition+by+both+caspase-4+and+caspase-5.">NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5.</a> European Journal of Immunology. 45 (10), 2918-2926 (2015).</li><li>Cullen, S. P., Kearney, C. J., Clancy, D. M., Martin, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+activators+of+the+NLRP3+inflammasome+promote+IL-1&#946;+secretion+by+triggering+necrosis.">Diverse activators of the NLRP3 inflammasome promote IL-1&#946; secretion by triggering necrosis.</a> Cell Reports. 11 (10), 1535-1548 (2015).</li><li>Sanders, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+imaging+of+inflammatory+caspase+dimerization+reveals+differential+recruitment+to+inflammasomes.">Single-cell imaging of inflammatory caspase dimerization reveals differential recruitment to inflammasomes.</a> Cell Death &amp; Disease. 6, 1813 (2015).</li><li>Charendoff, C. I., Bouchier-Hayes, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lighting+up+the+pathways+to+caspase+activation+using+bimolecular+fluorescence+complementation.">Lighting up the pathways to caspase activation using bimolecular fluorescence complementation.</a> Journal of Visualized Experiments: JoVE. (133), e57316 (2018).</li><li>Bouchier-Hayes, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cytoplasmic+caspase-2+activation+by+induced+proximity.">Characterization of cytoplasmic caspase-2 activation by induced proximity.</a> Molecular Cell. 35 (6), 830-840 (2009).</li><li>Riedhammer, C., Halbritter, D., Weissert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+blood+mononuclear+cells:+Isolation,+Freezing,+thawing,+and+culture.">Peripheral blood mononuclear cells: Isolation, Freezing, thawing, and culture.</a> Methods in Molecular Biology. 1304, 53-61 (2016).</li><li>Betsou, F., Gaignaux, A., Ammerlaan, W., Norris, P. J., Stone, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biospecimen+science+of+blood+for+peripheral+blood+mononuclear+cell+(PBMC)+functional+applications.">Biospecimen science of blood for peripheral blood mononuclear cell (PBMC) functional applications.</a> Current Pathobiology Reports. 7 (2), 17-27 (2019).</li><li>Perregaux, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-1+beta+maturation:+evidence+that+mature+cytokine+formation+can+be+induced+specifically+by+nigericin.">IL-1 beta maturation: evidence that mature cytokine formation can be induced specifically by nigericin.</a> The Journal of Immunology. 149 (4), 1294-1303 (1992).</li><li>Cheneval, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+mature+interleukin-1&#946;+(IL-1&#946;)+secretion+from+THP-1+cells+induced+by+nigericin+is+a+result+of+activation+of+p45+IL-1&#946;-converting+enzyme+processing.">Increased mature interleukin-1&#946; (IL-1&#946;) secretion from THP-1 cells induced by nigericin is a result of activation of p45 IL-1&#946;-converting enzyme processing.</a> Journal of Biological Chemistry. 273 (28), 17846-17851 (1998).</li><li>Fernandes-Alnemri, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pyroptosome:+a+supramolecular+assembly+of+ASC+dimers+mediating+inflammatory+cell+death+via+caspase-1+activation.">The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation.</a> Cell Death and Differentiation. 14 (9), 1590-1604 (2007).</li><li>Lu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unified+polymerization+mechanism+for+the+assembly+of+ASC-dependent+inflammasomes.">Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes.</a> Cell. 156 (6), 1193-1206 (2014).</li><li>Muller-Eberhard, U., Javid, J., Liem, H. H., Hanstein, A., Hanna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+report:+Plasma+concentrations+of+hemopexin,+haptoglobin+and+heme+in+patients+with+various+hemolytic+diseases.">Brief report: Plasma concentrations of hemopexin, haptoglobin and heme in patients with various hemolytic diseases.</a> Blood. 32 (5), 811-815 (1968).</li><li>Shyu, Y. J., Liu, H., Deng, X., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+new+fluorescent+protein+fragments+for+bimolecular+fluorescence+complementation+analysis+under+physiological+conditions.">Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions.</a> Biotechniques. 40 (1), 61-66 (2006).</li><li>Thornberry, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+heterodimeric+cysteine+protease+is+required+for+interleukin-1&#946;processing+in+monocytes.">A novel heterodimeric cysteine protease is required for interleukin-1&#946;processing in monocytes.</a> Nature. 356 (6372), 768-774 (1992).</li><li>Jensen, K., Anderson, J. A., Glass, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+small+interfering+RNA+(siRNA)+delivery+into+bovine+monocyte-derived+macrophages+by+transfection+and+electroporation.">Comparison of small interfering RNA (siRNA) delivery into bovine monocyte-derived macrophages by transfection and electroporation.</a> Veterinary Immunology and Immunopathology. 158 (3-4), 224-232 (2014).</li><li>Tada, Y., Sakamoto, M., Fujimura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+introduction+into+rice+by+electroporation+and+analysis+of+transgenic+plants:+use+of+electroporation+buffer+lacking+chloride+ions.">Efficient gene introduction into rice by electroporation and analysis of transgenic plants: use of electroporation buffer lacking chloride ions.</a> Theoretical and Applied Genetics. 80 (4), 475-480 (1990).</li><li>Bhowmik, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mutagenesis+in+wheat+microspores+using+CRISPR/Cas9.">Targeted mutagenesis in wheat microspores using CRISPR/Cas9.</a> Scientific Reports. 8 (1), 6502 (2018).</li><li>Sansom, D. M., Manzotti, C. N., Zheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What's+the+difference+between+CD80+and+CD86.">What's the difference between CD80 and CD86.</a> Trends in Immunology. 24 (6), 314-319 (2003).</li><li>Kurokawa, M., Kornbluth, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspases+and+kinases+in+a+death+grip.">Caspases and kinases in a death grip.</a> Cell. 138 (5), 838-854 (2009).</li></ol>

This protocol describes the workflow to obtain macrophages from monocytes isolated from human blood samples and a method to efficiently introduce the inflammatory caspase BiFC reporters into human MDM without compromising cell viability and behavior.
This protocol takes advantage of the BiFC technique35 to tag the inflammatory caspases at the caspase recruitment domain (CARD) with non-fluorescent fragments of the split fluorescent protein Venus. Inflammatory caspases en...

The caspases are a family of cysteine aspartate proteases that can be grouped into initiator caspases and executioner caspases. Executioner caspases comprise caspase-3, -6 and -7. They are naturally found in cells as dimers and are cleaved by the initiator caspases to execute apoptosis1. Initiator caspases include human caspase-1, -2, -4, -5, -8, -9, -10 and -12. They are found as inactive zymogens (pro-caspases) that are activated by proximity-induced dimerization and stabilized by auto-proteolytic cleavage2,3. The inflammatory caspases are a subset of the initiator caspases2 and encompass caspase-1, -4, -5, and -12 in humans, and caspase-1, -11, and -12 in mouse4,5. Rather than an apoptotic role, they play a central role in inflammation. They mediate proteolytic processing and secretion of pro-interleukin (IL)-1&#946; and pro-IL-186,7, which are the first cytokines to be released in response to pathogenic invaders8,9. Caspase-1 is activated upon recruitment to its activation platform; a large molecular weight protein complex termed the inflammasome (Figure 1A)10. Dimerization of caspase-4, -5, and -11 occurs independently of these platforms through a noncanonical inflammasome pathway11,12.
Canonical inflammasomes are cytosolic multimeric protein complexes that consist of an inflammasome sensor protein, the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), and the effector protein caspase-110. The most well studied canonical inflammasomes are the NOD-like receptor family containing a pyrin domain (NLRP), NLRP1 and NLRP3, the NLR family containing a CARD (NLRC), NLRC4, and the absent in melanoma 2 (AIM2). They each contain a pyrin domain, a CARD, or both domains. The CARD domain mediates the interaction between CARD-containing caspases and their upstream activators. Therefore, the scaffold molecule ASC, which is composed of an N-terminal pyrin domain (PYD) and a C-terminal CARD motif13,14, is required for recruitment of caspase-1 to the NLRP110, NLRP315, and AIM216 inflammasomes.
Each inflammasome is named after its unique sensor protein that recognizes distinct pro-inflammatory stimuli (Figure 1B). Activators of this pathway are termed canonical stimuli. Inflammasomes serve as sensors for microbial components and tissue stress, and assemble to trigger a robust inflammatory response through activation of the inflammatory caspases17. Inflammasome assembly initiates caspase-1 activation to mediate maturation and secretion of its main substrates pro-IL-1&#946; and pro-IL-18. This process occurs via a two-step mechanism. First, a priming stimulus upregulates the expression of certain inflammasome proteins and pro-IL-1&#946; through activation of the NF-&#954;B pathway. Second, an intracellular (canonical) stimulus induces inflammasome assembly and recruitment of procaspase-16,7.
Caspase-4 and caspase-5 are the human orthologs of murine caspase-1111. They are activated in an inflammasome-independent manner by intracellular lipopolysaccharide (LPS), a molecule found in the outer membrane of Gram-negative bacteria18,19,20, and by extracellular heme, a product of red blood cell hemolysis21. It has been proposed that LPS binds directly to the CARD motif of these proteins and induces their oligomerization20. Activation of caspase-4 or caspase-5 promotes IL-1&#946; release by inducing an inflammatory form of cell death called pyroptosis through cleavage of the pore-forming protein gasdermin D (GSDMD)18,19. In addition, the efflux of potassium ions resulting from caspase-4 and GSDMD-mediated pyroptotic death induces activation of the NLRP3 inflammasome and subsequent activation of caspase-122,23. Therefore, caspase-4, -5, and -11 are considered intracellular sensors for LPS that are able to induce pyroptosis and caspase-1 activation in response to specific stimuli11,24.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63162/63162fig01.jpg" /> 
Figure 1: Inflammatory caspases and caspase-bimolecular fluorescence complementation (BiFC) assay. (A) Diagram showing the caspase-BiFC system, where two caspase-1 prodomains (C1-pro) linked to each non-fluorescent fragment of Venus (Venus-C or Venus-N) are recruited to the NLRP3 activation platform, forcing Venus to refold and fluoresce. This complex appears as a green spot under the microscope and serves as a readout for inflammatory caspase-induced proximity, which is the first step in initiator caspase activation. (B)&#160;Schematic showing the domain organization of inflammasome components and inflammatory caspases. <a href="https://www.jove.com/files/ftp_upload/63162/63162fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Measuring specific initiator caspases activation is difficult, and there are not many methods available to do so by imaging approaches. Caspase Bimolecular Fluorescence Complementation (BiFC) can be used to visualize inflammatory caspase activation directly in living cells (Figure 1A)25. This technique has been recently adapted for use in human monocyte-derived macrophages (MDM)21. Caspase BiFC measures the first step in inflammatory caspase activation, induced proximity to facilitate dimerization. Expression of plasmids encoding the CARD-containing caspase prodomain fused to non-fluorescent fragments of the photostable yellow fluorescent protein Venus (Venus-C [VC]) and Venus-N [VN]) are used. When the two caspase prodomains are recruited to their activation platform or undergo induced proximity, the two halves of Venus are brought in close proximity and forced to refold and fluoresce (see Figure 1A,B). This provides a real-time readout of specific inflammatory caspase activation.
Human MDM abundantly express inflammasome genes and pattern recognition receptors that identify danger signals and pathogen products. This provides an ideal cell type for the interrogation of inflammatory caspase pathways. In addition, they can be derived from peripheral blood and even from patient samples to assess inflammatory caspase activation in a specific disease state. This protocol describes how to introduce the BiFC caspase reporters into MDM using nucleofection, an electroporation-based transfection method, how to treat the cells to induce inflammatory caspase activation, and how to visualize the active caspase complexes using microscopy approaches. Additionally, this methodology can be adapted to determine the molecular composition of these complexes, subcellular localization, kinetics, and size of these highly ordered structures25,26,27.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>48 well tissue culture2:34 plates</td>
			<td>Genesee Scientific</td>
			<td>25-108</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Tissue Culture Dishes</td>
			<td>VWR</td>
			<td>25382-166</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Mercaptoethanol 1000x</td>
			<td>Thermo Fisher Scientific</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>8 well chambered coverglass with 1.5 HP coverglass</td>
			<td>Cellvis</td>
			<td>c8-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoMACS columns</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-021-101</td>
			<td>For automated separation using AutoMACS Pro Separator only</td>
		</tr>
		<tr>
			<td>AutoMACS Pro Separator</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-092-545</td>
			<td>For automated separation using AutoMACS Pro Separator only</td>
		</tr>
		<tr>
			<td>AutoMACS Pro Washing Solution</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-092-987</td>
			<td>For automated separation using AutoMACS Pro Separator only</td>
		</tr>
		<tr>
			<td>AutoMACS Rinsing Solution</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-091-222</td>
			<td>For automated separation using AutoMACS Pro Separator only</td>
		</tr>
		<tr>
			<td>AutoMacs running buffer</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-091-221</td>
			<td>For manual or automated separation using QuadroMACS or AutoMACS pro Separator</td>
		</tr>
		<tr>
			<td>Axio Observer Z1 motorized inverted microscope equipped with a CSU-X1A 5000 spinning disk unit</td>
			<td>Zeiss</td>
			<td></td>
			<td>Any confocal microscope equipped with a laser module fitted with laser lines of 568 nm (RFP) and 488 or 512 nm (GFP or YFP) wavelengths can be used</td>
		</tr>
		<tr>
			<td>AxioObserver A1, Research Grade Inverted Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Any epifluorescence microscope with fluorescence filters capable of exciting 568 nm (RFP) and 488 or 512 nm (GFP or YFP) wavelengths can be used</td>
		</tr>
		<tr>
			<td>CD14+ MICROBEADS</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-050-201</td>
			<td>For manual or automated separation using QuadroMACS or AutoMACS pro Separator</td>
		</tr>
		<tr>
			<td>DPBS without calcium chloride and magnesium chloride</td>
			<td>Sigma</td>
			<td>D8537-6x500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DsRed mito plasmid</td>
			<td>Clontech</td>
			<td>632421</td>
			<td>Similar plasmids that can be used as fluorescent reporters can be found on Addgene</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS 6 x 100 mL</td>
			<td>Sigma</td>
			<td>GE17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement (100x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050079</td>
			<td></td>
		</tr>
		<tr>
			<td>GM-CSF</td>
			<td>Thermo Fisher Scientific</td>
			<td>PHC2011</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemin BioXtra, from Porcine, &#8805;96.0% (HPLC)</td>
			<td>Sigma</td>
			<td>51280-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630106</td>
			<td></td>
		</tr>
		<tr>
			<td>Inflammatory caspase BiFC plasmids</td>
			<td></td>
			<td></td>
			<td>Available by request from LBH lab</td>
		</tr>
		<tr>
			<td>LPS-EB Ultrapure</td>
			<td>Invivogen</td>
			<td>TLRL-3PELPS</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-042-401</td>
			<td>For manual separation using QuadroMACS Separator only</td>
		</tr>
		<tr>
			<td>MACS 15 mL Tube Rack</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-091-052</td>
			<td>For manual separation using QuadroMACS Separator only</td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-042-303</td>
			<td>For manual separation using QuadroMACS Separator only</td>
		</tr>
		<tr>
			<td>mCherry plasmid</td>
			<td>Yungpeng Wang Lab</td>
			<td></td>
			<td>Similar plasmids that can be used as fluorescent reporters can be found on Addgene</td>
		</tr>
		<tr>
			<td>Neon Transfection System</td>
			<td>Thermo Fisher Scientific</td>
			<td>MPK5000</td>
			<td>Includes Neon electroporation device, pipette and pipette station</td>
		</tr>
		<tr>
			<td>Neon Transfection System 10 &#181;L Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>MPK1096</td>
			<td>Includes resuspension buffer R, resuspension buffer T, electrolytic buffer E, 96 x 10 &#181;L Neon tips and Neon electroporation tubes</td>
		</tr>
		<tr>
			<td>Nigericin sodium salt, ready made solution</td>
			<td>Sigma</td>
			<td>SML1779-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine Hydrobromide</td>
			<td>Sigma</td>
			<td>P7280-5mg</td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS Separator</td>
			<td>Miltenyi (Biotec)</td>
			<td>130-090-976</td>
			<td>For manual separation using QuadroMACS Separator only</td>
		</tr>
		<tr>
			<td>qVD-OPh</td>
			<td>Fisher (ApexBio)</td>
			<td>50-101-3172</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875119</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5 M EDTA, pH 8.0</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Zen 2.6 (blue edition) software</td>
			<td>Zeiss</td>
			<td></td>
			<td>Any software used to operate the confocal microscope of choice</td>
		</tr>
	</tbody>
</table>

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.

The scheme shown in Figure 2 gives an overview of how to obtain, transfect, and image human MDM. After incubation of the selected CD14+ monocytes with GM-CSF for 7 days, the cell morphology changes over the course of the differentiation period (Figure 3A), going from spherical suspension cells to spindly and fully attached (days 3 and 4), and lastly to more spread cells when fully differentiated (day 7). Fully differentiated cells are then detached from the plat...

Watch this Scientific Journal Video about Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages at JoVE.com

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

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 ...

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