Name: an in vitro co-infection model to study plasmodium falciparum-hiv-1 interactions in human primary monocyte-derived immune cells
Uploaded: 2012-08-15T13:00:00
Duration: 7 min 39 s
Description: Watch this Scientific Journal Video about An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells at JoVE.com

This work was supported by the Canadian Institutes of Health Research through a Catalyst Co-infections and Co-morbidities grant. Human erythrocytes were provided by the Centre Hospitalier de l'Universit&eacute; Laval blood bank.

Note: Experiments with HIV-1 and Plasmodium falciparum must be performed in the proper biosafety level laboratories (BSL2 for parasites, and BSL3 for HIV-1 and co-infections) and special precaution must be taken when using potentially infected human blood.
1. Peripheral Blood Mononuclear Cells (PBMC) Purification (Based on 8 and 9)
<ol>
 <li>Start with fresh human blood from healthy donors (500 ml) treated with anticoagulants (heparin, citrate, acid citrate dextrose or citrate phosphate dextrose). Use endotoxin-free reagents when purifying and isolating PBMC and throughout the rest of the protocol.</li>
 <li>Disinfect the blood bag, including the tube, with 70% ethanol, cut the tube and carefully transfer the blood into a T150 flask.</li>
 <li>Prepare 16 tubes with 15 ml Lymphocytes Separation Medium (Ficoll) per 50 ml conical tube (make sure that Ficoll has reached room temperature). Carefully layer 30 ml of fresh blood on top.</li>
 <li>Centrifuge at 400 x g for 30 min at 20 &deg;C with breaks off.</li>
 <li>During the centrifugation, prepare 8 x 50 ml conical tubes with 25 ml of room temperature Hank's Balanced Salt Solution (HBSS) per tube.</li>
 <li>Carefully transfer the cloudy cell ring at the interface (contains PBMC) to the 50 ml tube containing the HBSS (pool 2 cell rings per tube). Complete volumes up to 50 ml with HBSS.</li>
 <li>Centrifuge at 150 x g for 15 min at 20 &deg;C with breaks on.</li>
 <li>During centrifugation of the cloudy cell ring, collect the yellow upper layer from the Ficoll step, pool the plasma to fill up a 50 ml conical tube. Inactivate complement in plasma by heating the 50 ml tube at 56 &deg;C for 30 min and spin 10 min at 1,800 x g. Keep the supernatant (contains diluted autologous plasma that can be used as medium supplement at later steps).</li>
 <li>Carefully remove the supernatant and resuspend the cell pellet from step 1.7 in 25 ml of HBSS and pool 2 pellets per 50 ml tube. Spin at 300 x g for 8 min at 20 &deg;C.</li>
 <li>Remove the supernatant carefully and resuspend the pellets in 10 ml HBSS and pool in one 50 ml tube.</li>
 <li>Take an aliquot, perform trypan blue exclusion to assess mortality and then count PBMC.</li>
 <li>Complete volume to 50 ml with HBSS and spin 10 min at 300 x g. Remove the supernatant and resuspend the cells in RPMI 1640 at 5x106 cells/ml (one should obtain around 400-500 x 106 PBMC from 500 ml of blood).</li>
</ol>
2. Monocytes-Derived Macrophages (MDM) Differentiation (Based on 8 and 9)
<ol>
 <li>Seed approximately 1.25 x 108 PBMC in 15 cm dishes with RPMI 1640 (25 ml/dish).</li>
 <li>Let cells adhere for 1-2 hr at 37 &deg;C with 5% CO2 atmosphere.</li>
 <li>Transfer non-adhered cells in a new flask and wash plate with 15 ml endotoxin-free PBS twice (5 min, 300 x g). The adhered cells are isolated monocytes.</li>
 <li>To allow freshly isolated monocytes to differentiate into MDM, add 20 ml of RPMI 1640 medium supplemented with 5% autologous plasma (from step 1.8). Add human recombinant Macrophage Colony Stimulating Factor (M-CSF, 25 ng/ml) and Penicillin/Streptomycin solution (PS, 100 U/ml Penicillin, 100 &mu;g/ml Streptomycin). Incubate for 2 days, change medium and incubate 2-3 extra days at 37 &deg;C in 5% CO2 atmosphere.</li>
 <li>Remove old medium and wash with endotoxin-free PBS once, add the PBS gently and aspirate immediately. To detach fully differentiated MDM, treat cells with ~7 ml Accutase (Accutase, a commercially available mix of proteases and collagenolytic activity that allow the detachment of cells from the plastic dish) for 15-20 min at 37 &deg;C in a 5% CO2 atmosphere, rock gently at mid-time.</li>
 <li>Add 3-5 ml autologous plasma (from step 1.8) in each plate (to help protect the cells while scraping).</li>
 <li>Gently scrape cells making sure that medium covers the cells at all times and transfer/pool in a 50 ml tube.</li>
 <li>Rinse plates with endotoxin-free PBS to recover as many cells as possible.</li>
 <li>Spin 5 min at 200 x g, discard supernatant.</li>
 <li>Resuspend pellet in 5 ml MDM culture medium (RPMI 1640 supplemented with 10% complement-inactivated Fetal Bovine Serum (iFBS), PS, 25 mM HEPES) and count cells (MDM yield usually 2-8% of total PBMC). Note: an aliquot of MDM might be taken in this step to assess cell population purity by flow cytometry; usually 99% of obtained MDM express CD14.</li>
 <li>Seed 1x105 MDM in 600 &mu;l of MDM culture medium per well in 24-well plates.</li>
 <li>Incubate overnight at 37 &deg;C in a 5% CO2 atmosphere before infections.</li>
</ol>
3. Production and Quantitation of HIV-1 Viral Stocks
<ol>
 <li>Produce viral stocks using calcium phosphate transfection in HEK293T cells following the protocol of Fortin et al. 10.</li>
 <li>In order to obtain single cycle viruses containing the luciferase-encoding reporter gene, co-transfect HEK293T cells with either 20 &mu;g of NL4.3Luc+Env-R+ and 10 &mu;g of pHCMV-G, or 15 &mu;g of NL4.3Luc+Env-R+ and 15 &mu;g of pJRFLenv. Use 30 &mu;g of NL4.3Luc+Env-R+ to generate control glycoprotein-deficient viruses. For the fully replicative virus, use 30 &mu;g of NL4.3Balenv. The vectors pHCMV-G and pJRFLenv encode the envelope glycoproteins of vesicular stomatitis virus (VSV-G) and of a CXCR5-tropic HIV-1 (different than NL4.3Balenv), respectively. Additional information on the plasmids can be obtained from 8. Plasmids can be obtained through the NIH AIDS Research and Reference Program (NIAID, NIH).</li>
 <li>Quantify all viral stocks using ELISA for the HIV-1 p24 capsid protein11, and verify their infectious potential prior to use. We assess the infectivity of our virus stocks through the use of TZM-bl indicator cells12.</li>
</ol>
4. Culture of Plasmodium falciparum Parasites (Based on 13)
4.1 General culture and maintenance of parasites
<ol>
 <li>Maintain P. falciparum 3D7 asexual stage parasites in human erythrocytes (blood group O+) at a 4% hematocrit in RPMI 1640 (buffered with 25 mM HEPES and 25 mM NaHCO3) supplemented with 0.5% (w/v) Albumax II, 25 &mu;g/ml Gentamicin and 370&mu;M Hypoxanthine at 37 &deg;C in a gas mixture of 1% oxygen/5% carbon dioxide in nitrogen.</li>
 <li>Parasitemia can be monitored by making a thin smear of the culture and staining with a 15% Giemsa solution.</li>
 <li>Always maintain an uninfected red blood cells (uRBC) culture dish in the same conditions as parasites to use as a control. 
 To obtain late stage parasites (trophozoites/early schizonts), synchronize the parasites as follows:</li>
</ol>
4.2 Parasite synchronization
<ol>
 <li>In the morning, harvest the parasite culture, spin 5 min at 300 x g and discard the supernatant.</li>
 <li>Resuspend pellet in 5 packed cell volumes with 5% (w/v) D-Sorbitol and incubate 5 min at 37 &deg;C.</li>
 <li>Spin 5 min at 300 x g, discard supernatant, resuspend pellet in 30 ml culture medium and put back in incubator.</li>
 <li>Approximately 8 hr later, repeat steps 4.2.1 to 4.2.3 in order to obtain tightly synchronized parasites. Late stage parasites (trophozoites/early schizonts) can then be harvested the following morning to perform the experiment. Prior to purification, perform Giemsa staining to confirm lifecycle stage.</li>
</ol>
4.3 Plasmodium falciparum-infected red blood cells (iRBC) purification
<ol>
 <li>Sterilize the VarioMacs separator (stand and magnet) with 70% ethanol and place it under the biological hood.</li>
 <li>Fix the three-way stopcock to CS column bottom.</li>
 <li>Cut the end of the needle plastic protector with the Miltenyi special cutter and fix the needle to the stopcock bottom.</li>
 <li>Lock the column into the magnet carefully.</li>
 <li>Remove all air bubbles in the column by slowly injecting 10 ml of MDM culture medium with the kit-enclosed syringe screwed on the left side of the stopcock.</li>
 <li>Wash column with ~20 ml MDM culture medium.</li>
 <li>Harvest RBC from the culture plate, wash once with 10 ml MDM medium (300 x g, 5 min) and resuspend the RBC pellet in 12 ml MDM culture medium. Slowly add to column and let the suspension flow through (only infected red blood cells containing parasites in the trophozoite or schizont stage will be retained by the magnetic field due to the presence of hemozoin crystals).</li>
 <li>Wash once with 20 ml medium and stop liquid flow when it reaches the white disc on top of the column.</li>
 <li>Remove the column from the magnet and elute iRBC in a clean sterile tube with 12 ml MDM culture medium.</li>
 <li>Smear an aliquot of the recovered iRBC and perform Giemsa staining to confirm lifecycle stage.</li>
 <li>Count recovered iRBC using a haemocytometer (isolated RBC are 100% iRBC).</li>
 <li>Pellet cells (300 x g, 5 min), discard supernatant and treat cells with 500 &mu;l fresh AB+ human serum (opsonisation step14).</li>
 <li>Incubate for 30 min at 37 &deg;C in a 5% CO2 atmosphere.</li>
 <li>Wash once with 10 ml MDM culture medium (300 x g, 5 min), and resuspend iRBC to desired concentration. Usually, a 10% parasitemia 15 cm culture dish roughly gives ~0.5-1x108 tightly synchronized iRBC.</li>
</ol>
5. Exposure of MDM to Plasmodium falciparum
<ol>
 <li>To expose the MDM to uRBC or iRBC, cells must be resuspended in MDM culture medium in order to obtain a ratio of uRBC/iRBC:MDM of 75:1. In previously prepared 24-well plates containing MDM, add appropriate RBC suspension volume to each well. Perform all experiments in triplicate.</li>
 <li>Incubate co-cultures for 4 hr at 37 &deg;C in a 5% CO2 atmosphere.</li>
 <li>Wash the cells with 600 &mu;l of endotoxin-free PBS 3 times, add the PBS gently and aspirate immediately.</li>
 <li>To lyse the remaining RBC, wash the wells by adding 200 &mu;l of ice-cold sterile water for 20 sec and aspirate.</li>
 <li>Add 600 &mu;l MDM culture medium and incubate 24 hr at 37 &deg;C in a 5% CO2 atmosphere.</li>
</ol>
6. Infection of MDM with a Fully Replicative HIV-1
<ol>
 <li>In order to assess the effect of P. falciparum on HIV-1 replication in MDM, add, according to the scheme outlined in Figure 1A, 10 ng of p24 (in 300 &mu;l of MDM media) of NL4.3Balenv viruses in the appropriate wells; add only MDM medium in control wells.</li>
 <li>Incubate 2 hr at 37 &deg;C in a 5% CO2 atmosphere.</li>
 <li>Wash the cells with endotoxin-free PBS 3 times, add the PBS gently and aspirate immediately. Add 600 &mu;l of fresh MDM medium per well.</li>
 <li>Incubate at 37 &deg;C in a 5% CO2 atmosphere.</li>
 <li>At days 3, 6, 9 and 12 following the start of viral infection, harvest 200 &mu;l of each supernatant, adding 200 &mu;l of fresh MDM medium thereafter. Keep the harvested supernatants at -20 &deg;C for quantitation of the major HIV-1 capsid p24 protein by ELISA following Fortin et al.10.</li>
</ol>
7. Infection of MDM with Single Cycle Virus Encoding Luciferase
<ol>
 <li>In order to determine the parasite's impact on HIV-1 gene expression in MDM, add, according to the scheme outlined in Figure 1B, 10 ng p24 (in 300 &mu;l of MDM medium) of either NL4.3Luc+Env-R+(VSV-G), NL4.3Luc+Env-R+(JRFLenv) or NL4.3Luc+Env-R+ viruses in the corresponding wells.</li>
 <li>Incubate 2 hr at 37 &deg;C in a 5% CO2 atmosphere.</li>
 <li>Wash the cells with 600 &mu;l of endotoxin-free PBS 3 times, add the PBS gently and aspirate immediately. Add 600 &mu;l of fresh MDM medium per well.</li>
 <li>Incubate at 37 &deg;C in a 5% CO2 atmosphere.</li>
 <li>Remove the medium 72 hr following infection and add 200 &mu;l of lysis buffer 1X (supplied with the luciferase assay kit). Let the cells lyse at room temperature with gentle shaking. Store at -20 &deg;C.</li>
 <li>Thaw the plate and transfer 40 &mu;l of the lysate to a 96-well luminometer plate; add 100 &mu;l of luciferase substrate (supplied with the luciferase assay kit) and measure the luciferase (Firefly luciferase) activity in a luminometer following the manufacturer's instructions.</li>
</ol>
8. Representative Results
Using our co-infection model, we show that exposure of P. falciparum to MDM decreases their susceptibility to HIV-1 infection. Indeed, a significant decrease (p&lt;0.05; 2 way ANOVA, day 12) in the release of viral particles, as measured by HIV-1 p24 capsid protein in the supernatant, is observed in MDM pretreated with parasites (Figure 2A). This observation is confirmed in cells infected by viruses encoding a luciferase-reporter gene. MDM infection with such viruses harboring either exogenous VSV-G or HIV-1 glycoproteins leads to significantly (p&lt;0.05, Student's t-test) less luciferase production in cells exposed to P. falciparum (Figure 2B). It is noteworthy that VSV-G-pseudotyped viruses yielded much greater luciferase activity than their JRFLenv counterparts; this is due to the greater infection efficiency of VSV-G pseudotyped particles15. Given that parasite exposition to MDM impacts both types of viruses, this suggests that it influences some step in viral gene expression (Figure 2B). It is also important to mention that cell viability was not affected by MDM exposition to iRBC (data not shown), indicating that the inhibition observed is specific and not due to cell mortality.
<img alt="Figure 1" src="/files/ftp_upload/4166/4166fig1.jpg" /> 
 Figure 1. A) Plate scheme for MDM infection with a fully replicative virus. Mock: uninfected cells. uRBC: cells exposed to uninfected red blood cells. iRBC: cells exposed to Plasmodium falciparum-infected red blood cells. Bal: cells infected with NL4.3Balenv. Bal/uRBC: cells exposed to uRBC and infected with NL4.3Balenv. Bal/iRBC: cells exposed to iRBC and infected with NL4.3Balenv. B) Plate scheme for MDM infection with single cycle luciferase-encoding virus. Mock: uninfected cells. uRBC: cells exposed to uninfected red blood cells. iRBC: cells exposed to Plasmodium falciparum-infected red blood cells. &Delta;env: cells infected with NL4.3Luc+Env-R+. JRFL: cells infected with NL4.3Luc+Env-R+(JRFLenv). vsv-g: cells infected with NL4.3Luc+Env-R+(vsv-g). &Delta;env/uRBC: cells exposed to uRBC and infected with NL4.3Luc+Env-R+. &Delta;env/iRBC: cells exposed to iRBC and infected with NL4.3Luc+Env-R+. JRFL/uRBC: cells exposed to uRBC and infected with NL4.3Luc+Env-R+(JRFLenv). JRFL/iRBC: cells exposed to iRBC and infected with NL4.3Luc+Env-R+(JRFLenv). vsv-g/uRBC: cells exposed to uRBC and infected with NL4.3Luc+Env-R+(vsv-g). vsv-g/iRBC: cells exposed to iRBC and infected with NL4.3Luc+Env-R+(vsv-g).
<img alt="Figure 2" src="/files/ftp_upload/4166/4166fig2.jpg" /> 
 Figure 2. A) Effect of Plasmodium falciparum on HIV-1 viral production in MDM. MDM were exposed to uRBC or iRBC at a ratio 75:1 (uRBC/iRBC:MDM) for 4 hr and extensively washed. Cells were infected with 10ng of NL4.3Balenv p24 for 2 hr. Viral production was monitored by ELISA for HIV-1 p24 in cell-free supernatant at different time points following initial viral infection. A representative experiment is shown. B) Effect of Plasmodium falciparum on HIV-1 viral transcription in MDM. MDM were infected with uRBC or iRBC at a ratio 75:1 (uRBC/iRBC:MDM). Cells were then infected with 10ng of p24 of single cycle virus (either NL4.3Luc+Env-R+, NL4.3Luc+Env-R+(JRFLenv) or NL4.3Luc+Env-R+(vsv-g)). Luciferase expression was evaluated in cell lysates 72 hr following initial virus infection. A representative experiment is shown.

<ol>
	<li>WHO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Malaria Report. , (2005).</li><li>Kublin, J. G., Patnaik, P., Jere, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Plasmodium+falciparum+malaria+on+concentration+of+HIV-1-RNA+in+the+blood+of+adults+in+rural+Malawi:+a+prospective+cohort+study.">Effect of Plasmodium falciparum malaria on concentration of HIV-1-RNA in the blood of adults in rural Malawi: a prospective cohort study.</a> Lancet. 365, 233-240 (2005).</li><li>Cuadros, D. F., Branscum, A. J., Crowley, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-malaria+co-infection:+effects+of+malaria+on+the+prevalence+of+HIV+in+East+sub-Saharan+Africa.">HIV-malaria co-infection: effects of malaria on the prevalence of HIV in East sub-Saharan Africa.</a> Int. J. Epidemiol. 40, 931-939 (2011).</li><li>Xiao, L., Owen, S. M., Rudolph, D. L., Lal, R. B., Lal, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+falciparum+antigen-induced+human+immunodeficiency+virus+type+1+replication+is+mediated+through+induction+of+tumor+necrosis+factor-alpha.">Plasmodium falciparum antigen-induced human immunodeficiency virus type 1 replication is mediated through induction of tumor necrosis factor-alpha.</a> J. Infect. Dis. 177, 437-445 (1998).</li><li>Froebel, K., Howard, W., Schafer, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+by+malaria+antigens+renders+mononuclear+cells+susceptible+to+HIV+infection+and+re-activates+replication+of+endogenous+HIV+in+cells+from+HIV-infected+adults.">Activation by malaria antigens renders mononuclear cells susceptible to HIV infection and re-activates replication of endogenous HIV in cells from HIV-infected adults.</a> Parasite Immunol. 26, 213-217 (2004).</li><li>Diou, J., Tardif, M. R., Barat, C., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+derived+from+hemozoin-loaded+monocytes+display+a+partial+maturation+phenotype+that+promotes+HIV-1+trans-infection+of+CD4++T+cells+and+virus+replication.">Dendritic cells derived from hemozoin-loaded monocytes display a partial maturation phenotype that promotes HIV-1 trans-infection of CD4+ T cells and virus replication.</a> J. Immunol. 184, 2899-2907 (2010).</li><li>Diou, J., Tardif, M. R., Barat, C., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malaria+hemozoin+modulates+susceptibility+of+immature+monocyte-derived+dendritic+cells+to+HIV-1+infection+by+inducing+a+mature-like+phenotype.">Malaria hemozoin modulates susceptibility of immature monocyte-derived dendritic cells to HIV-1 infection by inducing a mature-like phenotype.</a> Cell Microbiol. 12, 615-625 (2010).</li><li>Diou, J., Gauthier, S., Tardif, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ingestion+of+the+malaria+pigment+hemozoin+renders+human+macrophages+less+permissive+to+HIV-1+infection.">Ingestion of the malaria pigment hemozoin renders human macrophages less permissive to HIV-1 infection.</a> Virology. 395, 56-66 (2009).</li><li>Davies, J. Q., Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+human+macrophages.">Isolation and culture of human macrophages.</a> Methods Mol. Biol. 290, 105-116 (2005).</li><li>Fortin, J. F., Cantin, R., Lamontagne, G., Tremblay, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-derived+ICAM-1+glycoproteins+incorporated+on+human+immunodeficiency+virus+type+1+are+biologically+active+and+enhance+viral+infectivity.">Host-derived ICAM-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhance viral infectivity.</a> J. Virol. 71, 3588-3596 (1997).</li><li>Bounou, S., Leclerc, J. E., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+host+ICAM-1+in+laboratory+and+clinical+strains+of+human+immunodeficiency+virus+type+1+increases+virus+infectivity+and+CD4(+)-T-cell+depletion+in+human+lymphoid+tissue,+a+major+site+of+replication+in+vivo.">Presence of host ICAM-1 in laboratory and clinical strains of human immunodeficiency virus type 1 increases virus infectivity and CD4(+)-T-cell depletion in human lymphoid tissue, a major site of replication in vivo.</a> J. Virol. 76, 1004-1014 (2002).</li><li>Wei, X., Decker, J. M., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+resistant+human+immunodeficiency+virus+type+1+in+patients+receiving+fusion+inhibitor+(T-20)+monotherapy.">Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy.</a> Antimicrob. Agents Chemother. 46, 1896-1905 (2002).</li><li>Trager, W., Jensen, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+malaria+parasites+in+continuous+culture.">Human malaria parasites in continuous culture.</a> Science. 193, 673-675 (1976).</li><li>Turrini, F., Ginsburg, H., Bussolino, F., Pescarmona, G. P., Serra, M. V., Arese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis+of+Plasmodium+falciparum-infected+human+red+blood+cells+by+human+monocytes:+involvement+of+immune+and+nonimmune+determinants+and+dependence+on+parasite+developmental+stage.">Phagocytosis of Plasmodium falciparum-infected human red blood cells by human monocytes: involvement of immune and nonimmune determinants and dependence on parasite developmental stage.</a> Blood. 80, 801-808 (1992).</li><li>Akkina, R. K., Walton, R. M., Chen, M. L., Li, Q. X., Planelles, V., Chen, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+gene+transfer+into+CD34++cells+with+a+human+immunodeficiency+virus+type+1-based+retroviral+vector+pseudotyped+with+vesicular+stomatitis+virus+envelope+glycoprotein+G.">High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G.</a> J. Virol. 70, 2581-2585 (1996).</li><li>Zhao, C., Papadopoulou, B., Tremblay, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leishmania+infantum+enhances+human+immunodeficiency+virus+type-1+replication+in+primary+human+macrophages+through+a+complex+cytokine+network.">Leishmania infantum enhances human immunodeficiency virus type-1 replication in primary human macrophages through a complex cytokine network.</a> Clinical Immunol. 113, 81-88 (2004).</li></ol>

No conflicts of interest declared.

We have illustrated here two different approaches to analyze the impact of the malaria parasite on the HIV-1 viral cycle, i.e. by analyzing either viral gene expression or progeny virus production and replication in monocyte-derived macrophages. Similar approaches have been used for other HIV-1-parasite co-infections16. However, these new data are a step forward in the investigation of malaria-HIV-1 co-infections. Indeed, Diou et al.8 studied the effect of hemozoin, not live parasites, on HIV-1 replication; in agreement with our results, they observed that hemozoin was itself sufficient to inhibit viral production by MDM, and not MDMs.
Using the described experimental layout, we observed that P. falciparum exerts a clear detrimental effect on the HIV-1 replicative cycle in macrophages: a significant inhibition of viral production is observed in macrophages pre-exposed to parasites (Figure 2A) and a specific impact of the parasite on viral transcription is illustrated in Figure 2B. However, we cannot leave out any additional effects of the parasite on viral entry or fusion (decapsidation), or on post-integration mechanisms, such as protein synthesis or viral particle assembly and budding. Furthermore, it is possible that a different impact on HIV-1 replication would be obtained if the parasite were added either at the same time or following MDM infection with the virus.
Our in vitro co-infection model provides a powerful tool to perform detailed investigations of HIV-1/P. falciparum interactions in the host cell. For example, the combination of this experimental layout with other techniques such as quantitative real time PCR, which can target and quantify specific steps in viral retrotranscription and viral genome integration to the host cell genome, are quite feasible and should yield further insights into the mechanisms involved in co-infections. Moreover, specific assays to evaluate early steps of the viral cycle (viral fusion, decapsidation, entry quantitation) can be applied to this basic protocol to further analyze the effect on viral replication. These modifications show how flexible this protocol is concerning HIV-1 quantitation and detection: indeed, even standard HIV-1 reverse transcriptase quantitation assays using tritium-labeled nucleotides could conceivably replace the ELISA for HIV-1 p24. In addition to MDM, we expect our system to be adaptable to other cell types relevant for HIV-1-malaria interactions, such as monocytes and dendritic cells. The fact that MDM are adherent cells facilitates their manipulation, allowing for easy washing out of iRBC that have not been taken in, or that are in contact with MDM, without eliminating any macrophages. Such an advantage would not apply to dendritic cells and monocytes, which are cultured in suspension, complicating the separation of uRBC and free iRBC with such cells and we are currently addressing this issue. Our system could also be useful to study more complex interactions such as the effects of Plasmodium-exposed monocyte-derived cells on the HIV-1 viral cycle in other immune cells such as CD4-positive T cells in co-culture experiments. Finally, our protocol could be suitable for experiments looking at the effect of P. falciparum iRBCs on HIV-1 reactivation in PBMC for HIV-1 infected individuals.
Ethics statement
Human PBMC were obtained from the blood of healthy donors, in accordance with the guidelines of the Bioethics Committee of the Centre Hospitalier de l'Universit&eacute; Laval Research Center. A written consent was obtained from all blood donors.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>RPMI 1640</td>
 <td>Multicell</td>
 <td>350-000-CL</td>
 <td></td>
 </tr>
 <tr>
 <td>PBS-endotoxin free</td>
 <td>Sigma</td>
 <td>D8662 </td>
 <td></td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Wisent</td>
 <td>080-150 </td>
 <td>Heat-inactivated at 56 &deg;C for 30 min</td>
 </tr>
 <tr>
 <td>Accutase</td>
 <td>eBiosciences</td>
 <td>00-4555-56 </td>
 <td></td>
 </tr>
 <tr>
 <td>Albumax II</td>
 <td>Gibco</td>
 <td>11021-037</td>
 <td>Dissolved in RPMI 1640, filter-sterilized </td>
 </tr>
 <tr>
 <td>Lymphocyte separation medium</td>
 <td>Multicell</td>
 <td>305-010-CL</td>
 <td></td>
 </tr>
 <tr>
 <td>White luminometer plates</td>
 <td>Promega</td>
 <td>Z3291</td>
 <td></td>
 </tr>
 <tr>
 <td>CS Macs separation columms</td>
 <td>Miltenyi Biotech</td>
 <td>130-041-305 </td>
 <td></td>
 </tr>
 <tr>
 <td>VarioMacs separator</td>
 <td>Miltenyi Biotech</td>
 <td>130-090-282 </td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin solution</td>
 <td>Wisent</td>
 <td>450-201-EL </td>
 <td></td>
 </tr>
 <tr>
 <td>D-sorbitol</td>
 <td>Sigma-Aldrich</td>
 <td>S1876 </td>
 <td></td>
 </tr>
 <tr>
 <td>Cell scraper (25 cm)</td>
 <td>Sarstedt</td>
 <td>83.1830</td>
 <td></td>
 </tr>
 <tr>
 <td>24-Well Cell Culture Plates</td>
 <td>BD Falcon</td>
 <td>353047</td>
 <td></td>
 </tr>
 <tr>
 <td>150 x 20 mm culture dish</td>
 <td>Sarstedt</td>
 <td>83.1803.003</td>
 <td></td>
 </tr>
 <tr>
 <td>M-CSF</td>
 <td>Genscript</td>
 <td>Z02001</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H4034</td>
 <td></td>
 </tr>
 <tr>
 <td>Human serum AB+</td>
 <td>Valley Biomedical</td>
 <td>HP1022</td>
 <td></td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Wisent</td>
 <td>311-505-CL</td>
 <td></td>
 </tr>
 <tr>
 <td>Gentamicin</td>
 <td>Sigma-Aldrich</td>
 <td>G1397 </td>
 <td></td>
 </tr>
 <tr>
 <td>Hypoxanthine</td>
 <td>Sigma-Aldrich</td>
 <td>H9636</td>
 <td></td>
 </tr>
 <tr>
 <td>NaHCO3</td>
 <td>Sigma-Aldrich</td>
 <td>S5761</td>
 <td></td>
 </tr>
 <tr>
 <td>Luciferase Assay System</td>
 <td>Promega</td>
 <td>E1500</td>
 <td></td>
 </tr>
 <tr>
 <td>Human red blood cells</td>
 <td></td>
 <td></td>
 <td>Obtained purified from CHUL blood bank.</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells at JoVE.com

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.

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 ...

an-vitro-co-infection-model-to-study-plasmodium-falciparum-hiv-1

Research

JoVE Journal

Immunology and Infection

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

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 ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.