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.

<ol>
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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 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 ...

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

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

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 ...

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

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

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

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

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

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Quantification of Monocyte Chemotactic Activity In Vivo and Characterization of Blood Monocyte Derived Macrophages

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of monocyte-derived macrophages can impair wound healing. We showed that high fat and high sugar diets promote monocyte priming and dysfunction, converting healthy blood monocytes into a hyper chemotactic phenotype poised to differentiate into macrophages with dysregulated activation profiles and impaired phenotypic plasticity. The over-recruitment of monocyte-derived macrophages and recruitment of macrophages with dysregulated activation profiles is believed to be a major contributor to the development of chronic inflammatory diseases associated with metabolic disorders, including atherosclerosis and obesity. The goal of this protocol is to quantify the chemotactic activity of blood monocytes as a biomarker for monocyte priming and dysfunction and to characterize the macrophage phenotype blood monocytes are poised to differentiate into in these mouse models. Using single cell Western blot analysis, we show that after 24 h 33%of cells recruited into MCP-1-loaded basement membrane-derived gel plugs injected into mice are monocytes and macrophages; 58% after day 3. However, on day 5, monocyte and macrophage numbers were significantly decreased. Finally, we show that this assays also allows for the isolation of live macrophages from the surgically retrieved basement membrane-derived gel plugs, which can then be subjected to subsequent characterization by single cell Western blot analysis.

Here we present a protocol to quantify the chemotactic activity of blood monocytes in mouse models, to assess the effects of nutritional, pharmacological and genetic interventions on blood monocyte and to characterize the blood monocytes derived macrophages in mouse models using monocyte-chemoattractant protein-1 (MCP-1)-loaded basement membrane-derived gel plugs.

Tissue homeostasis and repair are critically dependent on the recruitment of monocyte-derived macrophages. Both under- and over-recruitment of ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

In Vitro Stimulation and Visualization of Extracellular Trap Release in Differentiated Human Monocyte-derived Macrophages

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic inflammation. Neutrophil ETs (NETs) consist of a mesh of DNA, histone proteins, and various granule proteins (i.e., myeloperoxidase, elastase, and cathepsin G). Other immune cells, including macrophages, can also produce ETs; however, to what extent this occurs in vivo and whether macrophage extracellular traps (METs) play a role in pathological mechanisms has not been examined in detail. To better understand the role of METs in inflammatory pathologies, a protocol was developed for visualizing MET release from primary human macrophages in vitro, which can also be exploited in immunofluorescence experiments. This allows further characterization of these structures and their comparison to ETs released from neutrophils. Human monocyte-derived macrophages (HMDM) produce METs upon exposure to different inflammatory stimuli following differentiation to the M1 pro-inflammatory phenotype. The release of METs can be visualized by microscopy using a green fluorescent nucleic acid stain that is impermeant to live cells (e.g., SYTOX green). Use of freshly isolated primary macrophages, such as HMDM, is advantageous in modeling in vivo inflammatory events that are relevant to potential clinical applications. This protocol can also be used to study MET release from human monocyte cell lines (e.g., THP-1) following differentiation into macrophages with phorbol myristate acetate or other macrophage cell lines (e.g., the murine macrophage-like J774A.1 cells).

Presented here is a protocol to detect macrophage extracellular trap (MET) production in live cell culture using microscopy and fluorescence staining. This protocol can be further extended to examine specific MET protein markers by immunofluorescence staining.

The release of extracellular traps (ETs) by neutrophils has been identified as a contributing factor to the development of diseases related to chronic ...

Determination of Vaccine Immunogenicity Using Bovine Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and play a unique role within the immune system by linking the innate and adaptive immune responses. These cells can phagocytize and then present captured antigens to effector immune cells, triggering a diverse range of immune responses. This paper demonstrates a standardized method for the in vitro generation of bovine monocyte-derived dendritic cells (MoDCs) isolated from cattle peripheral blood mononuclear cells (PBMCs) and their application in evaluating vaccine immunogenicity.
Magnetic-based cell sorting was used to isolate CD14+ monocytes from PBMCs, and the supplementation of complete culture medium with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to induce the differentiation of CD14+ monocytes into naive MoDCs. The generation of immature MoDCs was confirmed by detecting the expression of major histocompatibility complex II (MHC II), CD86, and CD40 cell surface markers. A commercially available rabies vaccine was used to pulse the immature MoDCs, which were subsequently co-cultured with naive lymphocytes.
The flow cytometry analysis of the antigen-pulsed MoDCs and lymphocyte co-culture revealed the stimulation of T lymphocyte proliferation through the expression of Ki-67, CD25, CD4, and CD8 markers. The analysis of the mRNA expression of IFN-&#947; and Ki-67, using quantitative PCR, showed that the MoDCs could induce the antigen-specific priming of lymphocytes in this in vitro co-culture system. Furthermore, IFN-&#947; secretion assessed using ELISA showed a significantly higher titer (**p &#60; 0.01) in the rabies vaccine-pulsed MoDC-lymphocyte co-culture than in the non-antigen-pulsed MoDC-lymphocyte co-culture. These results show the validity of this in vitro MoDC assay to measure vaccine immunogenicity, meaning this assay can be used to identify potential vaccine candidates for cattle before proceeding with in vivo trials, as well as in vaccine immunogenicity assessments of commercial vaccines.

The methodology describes the generation of bovine monocyte-derived dendritic cells (MoDCs) and their application for the in vitro evaluation of antigenic candidates during the development of potential veterinary vaccines in cattle.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) within the immune system. They patrol the organism looking for pathogens and ...