Name: visualization of inflammatory caspases induced proximity in human monocyte-derived macrophages
Uploaded: 2022-04-06T12:45:00.000+00:00
Duration: 8 min 41 s
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.

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The authors declare that they have no competing financial interests.

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 encode a catalytic domain composed of a large (p20) and a small (p10) subunit, and a conserved protein-protein interaction motif termed a CARD at their N-terminus or prodomain36 (Figure 1B). Recruitment of inflammatory caspases to their activation platforms is dependent on an intact CARD. For this approach, two caspase prodomain constructs (encoding the CARD motif) are generated, each linked to the non-fluorescent fragments of the photostable yellow protein Venus (Venus-C [VC]) and Venus-N [VN]) along with a fluorescent transfection reporter to allow visualization of the cells prior to or in the absence of activation of the caspase BiFC reporter. Fluorescence is observed only when interacting proteins come into close enough proximity that would allow for dimerization and activation (see Figure 1A). The efficiency of this process can be quantified using a standard epifluorescence or a confocal microscope. In combination with single-cell imaging, this protocol allows visualization of asynchronous events that may be undistinguishable in bulk populations, and depending on the resolution of the images, can be used to determine the size and localization of the BiFC complex associated with each caspase.
This protocol can be adapted to a range of proteins that are activated by oligomerization, and its applicability is not limited to microscopy methodologies. This approach also allows for the investigation of differential requirements for inflammatory caspases assembly. For example, siRNA can be used to identify upstream components/requirements for specific inflammatory caspase activation. The caspase-BiFC probes and the siRNA oligo (used to silence a target protein) can be transfected simultaneously into MDM. This can identify potential protein partners or essential components of each activation platform. For example, a siRNA oligo that targets the ASC adaptor molecule, which is required for assembly of the NLRP1, NLRP3, and AIM2 inflammasomes, was used to show that heme-induced activation of caspase-1 required these canonical inflammasomes while caspase-4 and -5 did not21. This methodology can also be adapted to time-lapse microscopy, so the kinetics of the inflammatory caspase activation can be determined in single cells. Using such an approach, one can assess when caspase dimerization occurs by precisely detecting the timing of appearance of the BiFC onset relative to the time when the cell undergoes death by monitoring loss of fluorescence of the transfection reporter (mCherry) as an indication of cell lysis. There are multiple ways to acquire and analyze caspase BiFC data; the choice will depend on the question to be answered and the kind of microscope and software available. For instance, single-time point data used to determine the efficiency of activation as well as time-lapse analysis can be especially effective if an auto-acquisition feature provided by some microscope software is available. This allows objective imaging, as predetermined positions arrays are acquired from each well of a multi-well plate. Images can be quantitated both by visual inspection and by using automated imaging software like CellProfiler, ImageJ, or MATLAB to increase accuracy and objectivity.
Investigating caspase pathways in primary cells is essential to fully understand the physiological roles of these proteins. In addition, because caspase-4 and caspase-5 are replaced by a single caspase-11 in mice, being able to assess human cells is essential. However, due to low transfection efficiencies and toxicities associated with many delivery systems in primary cells, it can be difficult to maximize the use of available plasmid-based reporters. The inflammatory caspase BiFC protocol describes a simple method to transfect MDM from peripheral blood of either patients or healthy subjects, reducing the number of cells required per transfection. This allows researchers to perform more complex experiments with precious cells like macrophages without compromising cell viability and cellular properties. The ability to use patient samples provides a major advantage of being able to assess inflammatory caspase activation in specific disease states. Indeed, with some minor optimizations, this protocol could be easily adapted to other murine or human primary cells.
This protocol uses an electroporation-based approach to transfect MDM. The Neon system is particularly advantageous since it does not induce an IFN-&#947; response in MDM, which could disrupt the specific immune response of the cells37. This is a small-scale electroporation system that uses a 10 &#181;L transfection volume with a high cell density to enable the efficient transfer of exogenous DNA to a large number of cells. It creates temporary pores in the cell membrane so that plasmids can pass quickly, avoiding the late endocytosis that occurs during chemical transfection and that can trigger plasmid degradation. This protocol uses an organic acid-based buffer formulation (buffer R) that introduces minimal cell toxicity when compared to other buffers with chloride ions38. The small volume and the design of the pipette tip chamber aid in generating a uniform electric field to maintain physiological conditions resulting in high survival rates. Transfection efficiency and cell survival are highly dependent on the chemical composition of the buffer, the cell density, the strength of the pulse, and the plasmid concentration39. Therefore, it is essential to optimize these conditions for each cell type and plasmid introduced. In MDM, lower voltages with longer pulse durations delivered plasmids more efficiently while keeping cells viable and healthy. Consequently, the settings used for this protocol were 1000 V and 2 pulses of 40 ms that resulted in transfection efficiencies between 40-60% with cell viability higher than 90% based on the ability of the cells to re-attach to the plate after transfection. While this protocol is optimized for human MDM, varying the voltage settings from 500 V to 1700 V, increasing the number of pulses from 1 to 3, and testing length of pulses ranging from 10 to 40 ms can balance transfection efficiency and cell viability in additional cell types and experimental settings.
There are several important considerations for optimal employment of the BiFC technique. 1) It is important that the user determines the optimal amount of each caspase plasmid to be introduced, as this may vary for different proteins and cell types. This is required to ensure maximal sensitivity and specificity of the assay. It is advised to run a pilot titration of the caspase BiFC plasmids (VC and VN fragments) between 200 ng and 1000 ng as shown in Figures 4A-B. The optimal range of plasmid introduced differs for each individual caspase, and therefore, they must be individually assessed. Choose a plasmid amount that provides the highest specific signal with minimal background. Ideally, the background level should be less than 10%, but up to 20% can be used if the specific signal is high. It is also critical to avoid harsh conditions and excessive manipulation of these cells during differentiation, transfection, and treatment. This can also result in spontaneous activation, and high background levels of caspase activation that can obscure the specific results since activation of immune cells such as monocyte or macrophages is a natural process, where they can acquire pro-inflammatory functions40. If using patient cells, it can be difficult to control for some inherent variation in levels of inflammasome activation and genetic background that can sensitize or desensitize them to stimuli and changes in their local microenvironment. For example, macrophage function can be affected in immune-compromised individuals or individuals fighting an infection, as this type of cell&#160;plays important homeostatic functions and are involved in the control of inflammation, among other functions. These genetic and environmental factors need to be considered when determining if a high background is non-specific or a biological effect. Using closely matched healthy controls (e.g., matched for gender and age) can help to assess the biological significance of a high background level of caspase BiFC. 2) It is critical that the VC and VN fragments are introduced at equal amounts since unequal expression of one Venus fragment could result in recruitment to the platform at higher frequencies and mask the full fluorescent signal, producing a false negative result. Therefore, the integrity and concentration of each individual plasmid should be validated before every transfection to avoid introducing different amounts of the VC and VN fragments. 3) Since this approach is based on the exogenous expression of the caspase reporters, additional controls for specificity should be included. Caspase-BiFC constructs containing single point mutations that disrupt binding between the caspase and its activation platform or silencing of one of the components of the activation platform using siRNA can be used to confirm that the fluorescent signal is due to specific binding of the caspase to the inflammasome. This control experiment should result in decreased levels of Venus-positive cells. 4) Expression of the caspase BiFC constructs could trigger or affect downstream events such as cell death. To circumvent this, non-enzymatic versions of the caspase are recommended for the BiFC reporters, such as the caspase prodomain or the full-length caspase where the catalytic cysteine residue is mutated and inactivated. To further control for this, control non-transfected treated and untreated wells should be included in the experiment and should show similar behavior to that of the transfected wells. 5) Endogenous caspase activity could possibly impact the BiFC readout. To control for this, one can express the caspase reporter in cells deficient for the caspase under study with the expectation that the BiFC efficiency would be the same.
One of the main drawbacks of this approach is that it does not measure caspase activation per se, but the induced proximity step required for activation to occur. This is a subtle but important distinction. By design, the prodomain does not contain the catalytic domains of the caspase that actually dimerize. Therefore, the refolding and fluorescence of the Venus fragments act as a proxy for caspase dimerization as they can only refold if the caspases are close enough to dimerize. Thus, the BiFC is specifically measuring induced proximity. It is possible that posttranslational modifications in the catalytic domains could impede dimerization without affecting the BiFC signal. For example, caspase-2 is phosphorylated on its catalytic domain, and this impedes dimerization without preventing the recruitment of caspase-2 to its activation platform41. Hence, observations using the BiFC approach must be complemented with functional studies to confirm that the fluorescence observed is representative of caspase activation. Despite this drawback, if the controls discussed here are included, the measurement of induced proximity by caspase BiFC can provide an accurate representation of specific caspase activation.
In conclusion, caspase BiFC is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level. This protocol allows for the interrogation of the inflammatory caspase signaling cascades in living immune cells. This approach not only provides a technique to specifically and accurately measure the first step in caspase activation but, by being applied to primary immune cells, has the potential to reveal properties of inflammatory caspases that could not be identified using conventional methods.

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.bdigitaluss.remotexs.co/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><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, &amp;ge;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 &amp;micro;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 &amp;micro;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 plate and transfected with the caspase BiFC pairs (VC and VN) along with the reporter plasmid (e.g., dsRedmito, a plasmid encoding a red fluorescent protein targeted to the mitochondria), which is used to label the transfected cells (Figure 3B) and employed to assess the efficiency of the transfection after 24 h. Figures 3B-D show an example of BiFC results using the caspase-1 pro BiFC transfected cells treated for 20 h with nigericin (5 &#181;M), a known pro-inflammatory stimulus that triggers the assembly of the NLRP3 inflammasome30,31. Untreated cells show no Venus fluorescence (Figure 3B), and in nigericin-treated cells, caspase-1 BiFC appears as a single punctum with the typical shape of ASC specks32,33,25 (Figure 3C). Figure 3D shows an example of quantitation of Venus-positive MDM. The highest percentage of caspase-1 BiFC is seen in the LPS + nigericin treatment group (Figure 3D). This result is consistent with activation of a canonical inflammasome, where both a priming signal (LPS) and an intracellular signal (nigericin) are required for activation of capase-1.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63162/63162fig03.jpg" /> 
Figure 3: Differentiation of CD14-monocytes and transfection of human MDM. (A) Representative bright-field images of CD14+ monocytes from peripheral blood exposed to GM-CSF at days 1, 3, 4, and 7 of differentiation (Scale bar, 100 &#181;m). (B-C) Human MDM were transfected with caspase-1 pro BiFC pairs and the transfection reporter dsRedmito (50 ng, red). 24 h later, cells were primed with and without LPS (100 ng/mL) for 3 h and treated with nigericin (5 &#181;M) in imaging medium for 20 h. Representative confocal images of untreated and nigericin-treated cells are shown (Scale bar, 10 &#181;m). The BiFC is shown in green. (D) Cells from (C) were assessed for the percentage of dsRedmito-positive cells (red, transfected cells) that were Venus-positive (green, caspase-1 BiFC complex) at 20 h. Error bars represent SD of at least two independent counts per well. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63162/63162fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
An example of plasmid titration is shown in Figure 4, where increasing amounts of each BiFC plasmid is transfected. This allows for the selection of the optimal dose of plasmid, resulting in a specific signal and minimal background. Figure 4A shows the results of human MDM transfected with increasing concentrations of the caspase-1, -4, and -5 pro BiFC pairs (VC and VN) treated with heme, a highly pro-inflammatory molecule that results from red blood cell destruction34. The highest percentage of Venus-positive cells for caspase-1, -4, and -5 pro BiFC pairs results from 400 ng, 500 ng, and 1000 ng of transfected plasmid, respectively. However, using the highest amount of plasmid also runs the risk of increasing non-specific background (Figure 4B). For example, transfection of 1000 ng of the caspase-5 pro BiFC pair results in almost 40% non-specific background. Therefore, using the lower 700 ng amount is considered optimal for this plasmid pair (Figure 4B). In Figure 4C, representative confocal images obtained with a 20x objective of a field of cells 24 h post-transfection are shown. In this image, one can see that the untreated transfected cells are viable based on the appearance of the mitochondria (mitochondria in dead cells are highly fragmented) and the morphology of the cells (apoptotic cells are shrunken). After treatment with heme, the caspase-1 complex appears as a single green punctum similar to the one induced by nigericin in Figure 3C, and its appearance was accompanied by cell shrinkage.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63162/63162fig04.jpg" /> 
Figure 4: Human MDM transfected with different amounts of inflammatory caspase pro BiFC pairs. (A) Titration of caspase-1 pro; caspase-4 pro; or caspase-5 pro BiFC pairs. Human MDM were transfected with the indicated amounts of the caspase pro BiFC pairs with dsRedmito as a transfection reporter (50 ng) and incubated overnight. On the next day, the culture medium was removed from all wells, and cells were treated with and without heme (50 &#181;M) in 0.1% FBS medium. After 1 h, an equal amount of complete medium was added to all wells to reconstitute the FBS concentration to 5% and prevent heme from killing the cells. After a total treatment of 20 h, cells were assessed for the percentage of dsRedmito-positive transfected cells that were Venus-positive, determined from a minimum of 300 cells per well. Error bars represent SD of at least three independent counts per well. (B)&#160;Human MDM were transfected with selected amounts of caspase-1 pro; caspase-4 pro; or caspase-5 pro BiFC pairs, along with dsRedmito (50 ng) as a reporter for transfection. 24 h post-transfection, cells were treated with or without heme (50 &#181;M) in 0.1% FBS. After 1 h, FBS was reconstituted to 5% to inhibit extracellular heme. Cells were assessed for the percentage of dsRedmito-positive cells (red, transfected cells) that were Venus-positive (green, caspase BiFC complex) at 20 h determined from a minimum of 300 cells per well. Results are represented as percentage Venus-positive cells. Error bars represent SD of at least three independent counts per well. (C) Representative confocal images obtained with a 20x objective of a field of cells 24 h post-transfection and treated with and without heme as described in (B). Red: dsRedmito-positive cells; Green: Venus-positive cells; Scale bar, 20 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63162/63162fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

visualization-inflammatory-caspases-induced-proximity-human-monocyte

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

2.6K Views.  Baylor College of Medicine. 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.

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

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

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

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 Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

Biology

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.

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 ...

Visualization of Caspase Activation in Human Monocyte-Derived Macrophages

Source: Bol&#237;var, B. E., et. al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/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.

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

Detection of Inflammasome Activation via Fluorescence Microscopy

Source: den Hartigh, A. B. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/57463">Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages.</a> J. Vis. Exp.&#160;(2018)
This video demonstrates a method to detect NLRP3 inflammasome activation in macrophages using fluorescent reporter probes. The cells are treated with potassium ionophores and glycine to induce inflammasome assembly and caspase-1 activation. Fluorescent reporter probes visualize activated caspase-1, and cells are stained with a fluorescent dye for nuclei visualization.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Host Defense Mechanism

An In Vitro Technique to Study Inflammasome Complex Formation in Macrophages

Source: den Hartigh, A. B., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/57463">Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages.</a> J. Vis. Exp. (2018).
This video demonstrates a technique to study NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome formation in primary macrophages via immunofluorescence. The macrophages are first primed with bacterial lipopolysaccharide (LPS) to upregulate NLRP3 protein expression. Exposing the macrophages to nigericin&#8212;a potassium ionophore&#8212;leads to the activation of NLRP3 and the formation of inflammasomes, which are visualized via immunofluorescence.

Assessment of the Activation of Multiple Caspases in Macrophages

Source: Han, J. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/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.

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

Summary

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Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

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

Visualization of Vascular Ca²⁺ Signaling Triggered by Paracrine Derived ROS

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Visualization of Caspase Activation in Human Monocyte-Derived Macrophages

Detection of Inflammasome Activation via Fluorescence Microscopy

An In Vitro Technique to Study Inflammasome Complex Formation in Macrophages

Assessment of the Activation of Multiple Caspases in Macrophages