This work was supported by the German Research Council (DFG) via the project STE 432/11-1 awarded to AS and by the ELAN Program from the Faculty of Medicine (Friedrich-Alexander-Universit&#228;t Erlangen-N&#252;rnberg) via the project 18-12-21-1, granted to LG.

Monocyte-derived DCs were generated from leukapheresis products of healthy donors. For this, a positive vote from the local ethics committee has been obtained (reference number 4556). The experiments of the present study were performed in accordance with the recommendations of the ethics committee of the &#34;Friedrich-Alexander-Universit&#228;t Erlangen-N&#252;rnberg&#34;&#160;(reference number 4556). All donors approved a written informed consent, including the accordance with the Declaration of Helsinki.
1. Generation and handling of immature dendritic cells (iDCs) and mature dendritic cells (mDCs)
<ol>
	<li>Isolate human peripheral blood mononuclear cells (PBMCs) from leukoreduction system chambers (LRSCs) as previously described27. Avoid cryopreservation of PBMCs and use them directly upon isolation to obtain higher DC yields.</li>
	<li>Generate human DCs from PBMCs of different healthy donors in T175 cell culture flasks as previously described10,27. Briefly, use 350-400 millions of PBMCs in 30 mL of DC medium (RPMI 1640 without L-glutamine, 1% (v/v) AB-serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 0.4 mM L-glutamine, 10 mM HEPES) per cell culture flask for isolation of monocytes by adherence. After 1 h, wash off nonadherent fraction using RPMI 1640. Add fresh DC medium supplemented with 800 U/mL GM-CSF and 250 U/mL IL-4, and incubate for 3 days.
	<ol>
		<li>On day 3 post adherence, add 5 mL of fresh DC medium containing GM-CSF and IL-4 with a final concentration of 400 U/mL and 250 U/mL per cell culture flask, respectively, for DC differentiation.</li>
		<li>To harvest iDCs, gently rinse loosely-adherent iDCs from the bottom of the cell culture flask, on day 4 post adherence. Repeat this step 2 times. For generation of mDCs, add maturation cocktail composed as follows: GM-CSF (final concentration: 40 U/mL), IL-4 (final concentration: 250 U/mL), IL-6 (final concentration: 1000 U/mL), IL-1&#946; (final concentration: 200&#160;U/mL), TNF-&#945; (final concentration: 10 ng/mL), prostaglandin E2 (PGE2; final concentration: 1&#160;&#956;g/mL).</li>
		<li>Six days post adherence (two days post induction of maturation using a cytokine cocktail), rinse mDCs from the bottom of the cell culture flask. Repeat this step two times. 
		NOTE: Immature and mature DCs can be sequentially generated from identical donors in 1 cell culture flask. To do so, (i) separate the appropriate number of iDCs and (ii) induce maturation of the remaining cells in the flasks using the cytokine cocktail listed in step 1.2.2.</li>
	</ol>
	</li>
	<li>Transfer iDCs or mDCs in the respective cell culture medium into 50 mL tubes. Harvest the cells via centrifugation at 300 x g for 5 min.
	<ol>
		<li>Gently resuspend (=wash) DCs in 5-10 mL of RPMI 1640 per cell culture flask. Combine respective DC suspensions in one tube.</li>
		<li>Define the cell number using a counting chamber or an alternative method. Avoid temperature alterations when handling iDCs, to reduce the risk of phenotypic changes.</li>
	</ol>
	</li>
</ol>
2. Flow cytometric analyses to monitor the phenotypic maturation status
<ol>
	<li>Transfer iDCs or mDCs (0.5 x 106) from step 1.3.1 into a 1.5 mL tube. Harvest the cells via centrifugation at 3390 x g for 1.5 min. Wash the cells once with FACS buffer (PBS supplemented with 2% fetal calf serum (FCS)).</li>
	<li>Resuspend the cells in 100 &#181;L of antibody staining solution (FACS buffer) containing specific fluorochrome-labeled antibodies against defined surface molecules.
	<ol>
		<li>Use the following antibodies to verify purity (CD3-FITC, CD14-PE) as well as maturation status of DCs (CD80-PacBlue/-PE-Cy5, CD11c-PE-Cy5, CCR7-PE-Cy7, CD83-APC, CD86-PE, MHCII-APC-Cy7).</li>
		<li>Prepare one unstained sample in 100 &#181;L of FACS buffer as a control.</li>
		<li>Stain the cells on ice in the dark for 30 min.</li>
	</ol>
	</li>
	<li>Subsequently wash the cells two times in 1 mL of FACS buffer and centrifuge at 3390 x g for 1.5&#160;min.</li>
	<li>Finally, resuspend the cells in 200 &#181;L of FACS buffer supplemented with 2% PFA and analyze the cells by flow cytometry. Fixed cells can be stored at 4&#176; C in the dark up to 2 days.</li>
</ol>
3. Infection procedure of DCs with Herpes simplex virus type-1 (HSV-1) and interference of HSV-1-induced autophagic flux via spautin-1 or bafilomycin-A1
NOTE: The strain HSV-1/17+/CMV-EGFP/UL43 (HSV-1 EGFP) used in this study was obtained from the laboratory strain HSV-1 strain 17+. The HSV-1 EGFP strain expresses the enhanced green fluorescent protein (EGFP) which has been inserted into the UL43 gene locus under control of the CMV promoter. EGFP serves as a marker for HSV-1 infection. Moreover, the strain HSV1-RFPVP26 was used for DC infection studies (previously described in Turan et al., 2019). This virus expresses the capsid surface protein VP26 fused to monomer red fluorescent protein (mRFP).
<ol>
	<li>Transfer iDCs or mDCs (2 x 106) from step 1.3 into a 2 mL tube. Subsequently, centrifuge the cells at 3390 x g for 1.5 min and discard the supernatant.</li>
	<li>Gently resuspend the cells in infection medium (RPMI 1640 supplemented with 20 mM HEPES).
	<ol>
		<li>To inhibit the autophagosomal-lysosomal degradation pathway, pre-treat DCs with spautin-1 or bafilomycin-A1 1 h prior to infection. Add either 10 &#181;M spautin-1 or 1 &#181;M BA1, or DMSO as untreated control, to the infection medium. Incubate the cells in a heating block at 300 rpm shaking at 37 &#176;C for 1 h.</li>
		<li>For infection studies, inoculate the cells with HSV-1 virions at a multiplicity of infection (MOI) of 2. Add the respective volume of MNT buffer (30 mM 2-(N-morpholino)ethanesulfonic acid (MES), 100 mM NaCl, 20 mM Tris) as mock control. Incubate the cells in a heating block at 300 rpm shaking at 37 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>1 h post infection (hpi), collect the cells at 3390 x g for 1.5 min. Aspirate inoculum and gently resuspend the cells in DC medium containing 40 U/mL of GM-CSF, 250 U/mL of IL-4 and either 10 &#181;M spautin-1, 1 &#181;M BA1, or DMSO as control. Seed mock-treated and HSV-1-infected cells at a final concentration of 1 x 106/mL into a 6-well plate.</li>
	<li>At 16-24 hpi, harvest the cells by rinsing (mDCs) or using a cell scraper (iDCs). Transfer the cells into a 1.5 mL safelock tube.
	<ol>
		<li>Collect the cells via centrifugation at 3390 x g for 1.5 min and wash the pellet once by adding 1 mL of PBS.</li>
		<li>Vigorously resuspend the cells in lysis mix containing 29 &#181;L of 2x Roti-Load, 1 &#181;L 100&#160;mM MgCl2 and 12.5 U/mL benzonase.</li>
		<li>For cell lysis and DNA digestion using benzonase, incubate the samples at 37 &#176;C for 10 min. Subsequently, denature the proteins at 95 &#176;C for 10 min.</li>
		<li>Perform SDS-PAGE and Western blot analyses to verify protein levels of LC3BI/II, p62, ICP0/5, and GAPDH.</li>
	</ol>
	</li>
</ol>
4. Interference of HSV-1-induced autophagic flux via electroporation of iDCs using FIP200-siRNA
NOTE: The present protocol for siRNA electroporation was modified from Prechtel et al. (2007) and Gerer et al. (2017).
<ol>
	<li>Transfer iDCs (12 x 106) at day 3.5 post adherence into a 50 mL tube. Subsequently, centrifuge the cells at 300 x g for 5 min and discard the supernatant. In parallel, perform flow cytometric analysis to monitor the maturation status as described in step 2 (Use Life/Dead violet instead of CD80-PacBlue).</li>
	<li>Gently wash iDCs in 5 mL of OptiMEM without phenol red and centrifuge the cells at 300 x g for 5 min. Discard the supernatant and gently resuspend iDCs in 200 &#181;L of OptiMEM without phenol red, adjusting a cell concentration of 6 x 106/100 &#181;L. Do not place the cells on ice and avoid temperature alterations. Move on quickly and avoid long incubation periods of iDCs in OptiMEM without phenol red.</li>
	<li>Transfer either 75&#160;pmol of FIP200-specific siRNA or 75 pmol of scrambled siRNA, as a control, into 4 mm electro cuvettes and add 100 &#181;L (6x106 cells) of the cell suspension.&#160;Directly pulse iDCs using the electroporation apparatus I, applying the following settings: 500 V for 1 ms.
	<ol>
		<li>Prior to the experimental procedure, prepare siRNA suspensions according to the manufacturer&#8217;s instruction, aliquot and store them at -20 &#176;C. Thaw and keep them on ice when using these siRNAs for electroporation. Before electroporating the samples, perform a test pulse.</li>
	</ol>
	</li>
	<li>After electroporation, directly transfer iDC into 6-well plates with fresh pre-warmed DC medium (supplemented with 40 U/mL of GM-CSF and 250 U/mL of IL-4). Seed the cells at a final concentration of 1 x 106/mL and place them into an incubator. Do not rinse the cells out of the electro cuvette.</li>
	<li>After 48 h, first examine the morphology of electroporated iDCs microscopically. Then, harvest the cells using a cell scrapper and transfer them into 15 mL tubes. Rinse the wells with 1 mL of PBS supplemented with 0.01% EDTA and transfer the solution in the respective tubes.</li>
	<li>Subsequently, split 6 x 106 iDCs per siRNA condition as described in the next steps.
	<ol>
		<li>Use 0.5 x 106 cells to assess the maturation status and cell viability as described in step 2. Use the following antibodies: CD11c-PE-Cy5, CCR7-PE-Cy7, CD83-APC, MHCII-APC-Cy7 and Life/Dead violet.</li>
		<li>Use 1 x 106 cells for Western blot analyses to verify FIP200-specific knockdown efficiency. Transfer and harvest the cells into a 1.5 mL safelock tube by centrifugation at 3390 x g for 1.5 min. Prepare cell lysates as described in step 3.4 and perform Western blot analyses.</li>
		<li>Use the remaining cells (4.5 x 106) for HSV-1 infection experiments. For each experimental condition, transfer 2.25 x 106 iDCs into 2 mL tubes and either infect them with HSV1 at an MOI of 2 or add MNT buffer as a mock control. Perform the infection as described in step 3.3.</li>
		<li>At 20 hpi, harvest the cells using a cell scraper and prepare cell lysates for Western blot analyses as described in step 3.4.</li>
	</ol>
	</li>
</ol>
5. Modulation of the autophagosome-lysosomal pathway in HSV-1-infected mDCs using KIF1B/2A-siRNA electroporation
<ol>
	<li>Transfer iDCs (24 x 106) at day 4 post adherence into a 50 mL tube. Subsequently, centrifuge the cells at 300 x g for 5 min and discard the supernatant.</li>
	<li>Gently resuspend (= wash) iDCs in 8 mL of PBS, to adjust a final cell concentration of 3 x 106/mL. Transfer 3 x 106 cells into 1.5 mL tubes and harvest the cells at 3390 x g for 1.5 min.</li>
	<li>Resuspend iDCs in 100 &#181;L of buffer P3 containing the supplement mix (according to the manufacturer&#8217;s instructions; electroporation kit apparatus II) and either (i) 75&#160;pmol of KIF1B-specific siRNA, (ii) 75 pmol of KIF2A-specific siRNA, or (iii) both. Use (iv) the respective amount of scrambled siRNA as a control. Prepare two tubes for each siRNA condition (6 x 106 cells) and transfer the suspensions into separate electro cuvettes. Directly pulse iDCs applying the pulse &#34;EH-100&#34;&#160;using the electroporation apparatus II.
	<ol>
		<li>Prior to the experimental procedure, prepare siRNA suspensions according to the manufacturer&#8217;s instruction, aliquot and store them at -20 &#176;C. Thaw and keep them on ice when using these siRNAs for electroporation. Do not place iDCs on ice and avoid temperature alterations. Move on quickly and avoid long incubation of iDCs in PBS or buffer P3.</li>
	</ol>
	</li>
	<li>Directly after electroporation, add 500 &#181;L of pre-warmed RPMI 1640 to electro cuvettes. Incubate the cells in an incubator for 5-10 min. Transfer iDCs into 6-well plates with fresh pre-warmed DC medium (supplemented with 40 U/mL of GM-CSF and 250 U/mL of IL-4). Combine respective conditions into one well, seed the cells at a final concentration of 1&#183;106/mL and place them into an incubator.</li>
	<li>4 h after incubation, add the maturation cocktail containing the cytokines listed in step 1.2.2. 
	NOTE: Prepare one sample from non-electroporated DCs (1 x 106) as control for flow cytometric analyses 2 days post electroporation. Treat control cells analogously to electroporated samples.</li>
	<li>Two days post electroporation, harvest cells by resuspension and transfer into 15 mL tubes. Rinse the wells with 1 mL of PBS and transfer the suspensions in the respective tubes. Split 6&#183;106 DCs per siRNA condition as described in the following steps:
	<ol>
		<li>Use 0.25 x 106 DCs (electroporated and non-electroporated) to check for maturation status and cell viability as described in step 2. Use the following antibodies: CD80-PE-Cy5, CD83-APC, CD86-PE, MHCII-APC-Cy7, and Life/Dead violet.</li>
		<li>Use 0.75 x 106 cells for Western blot analyses to assess KIF1B/2A-specific knockdown efficiency. Collect the cells into a 1.5 mL safelock tube by centrifugation at 3390 x g for 1.5 min. Prepare cell lysates as described in step 3.4 and perform Western blot analyses.</li>
		<li>Use the remaining cells (5 x 106) from each siRNA condition and perform HSV-1 infection experiments. For each experimental condition, transfer 2.5 x 106 iDCs into 2 mL tubes and either infect them with HSV-1 at an MOI of 2 or add MNT buffer as a mock control. Perform the infection as described in step 3.3.</li>
		<li>At 20 h post infection, harvest the cells by resuspension and prepare cell lysates for Western blot analyses to verify the induction of HSV-1-induced autophagic turnover, as described above in step 3.4.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Chapuis, F., Rosenzwajg, M., Yagello, M., Ekman, M., Biberfeld, P., Gluckman, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+dendritic+cells+from+monocytes+in+vitro.">Differentiation of human dendritic cells from monocytes in vitro.</a> European Journal of Immunology. 27 (2), 431-441 (1997).</li><li>Kummer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+type+1+induces+CD83+degradation+in+mature+dendritic+cells+with+immediate-early+kinetics+via+the+cellular+proteasome.">Herpes simplex virus type 1 induces CD83 degradation in mature dendritic cells with immediate-early kinetics via the cellular proteasome.</a> Journal of Virology. 81 (12), 6326-6338 (2007).</li><li>Kruse, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mature+dendritic+cells+infected+with+herpes+simplex+virus+type+1+exhibit+inhibited+T-cell+stimulatory+capacity.">Mature dendritic cells infected with herpes simplex virus type 1 exhibit inhibited T-cell stimulatory capacity.</a> Journal of Virology. 74 (15), 7127-7136 (2000).</li><li>Salio, M., Cella, M., Suter, M., Lanzavecchia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+dendritic+cell+maturation+by+herpes+simplex+virus.">Inhibition of dendritic cell maturation by herpes simplex virus.</a> European Journal of Immunology. 29 (10), 3245-3253 (1999).</li><li>Prechtel, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+mature+dendritic+cells+with+herpes+simplex+virus+type+1+dramatically+reduces+lymphoid+chemokine-mediated+migration.">Infection of mature dendritic cells with herpes simplex virus type 1 dramatically reduces lymphoid chemokine-mediated migration.</a> Journal of General Virology. 86, 1645-1657 (2005).</li><li>Theodoridis, A. A., Eich, C., Figdor, C. G., Steinkasserer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+dendritic+cells+with+herpes+simplex+virus+type+1+induces+rapid+degradation+of+CYTIP,+thereby+modulating+adhesion+and+migration.">Infection of dendritic cells with herpes simplex virus type 1 induces rapid degradation of CYTIP, thereby modulating adhesion and migration.</a> Blood. 118 (1), 107-115 (2011).</li><li>Cohrs, R. J., Gilden, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+herpesvirus+latency.">Human herpesvirus latency.</a> Brain Pathology. 11 (4), 465-474 (2001).</li><li>Grinde, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpesviruses:+latency+and+reactivation+-+viral+strategies+and+host+response.">Herpesviruses: latency and reactivation - viral strategies and host response.</a> Journal of Oral Microbiology. 5, (2013).</li><li>Whitley, R. J., Roizman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+infections.">Herpes simplex virus infections.</a> The Lancet. 357 (9267), 1513-1518 (2001).</li><li>Turan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagic+degradation+of+lamins+facilitates+the+nuclear+egress+of+herpes+simplex+virus+type+1.">Autophagic degradation of lamins facilitates the nuclear egress of herpes simplex virus type 1.</a> The Journal of Cell Biology. 218 (2), 508-523 (2019).</li><li>Takeshige, K., Baba, M., Tsuboi, S., Noda, T., Ohsumi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+yeast+demonstrated+with+proteinase-deficient+mutants+and+conditions+for+its+induction.">Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction.</a> The Journal of Cell Biology. 119 (2), 301-311 (1992).</li><li>Yin, Z., Pascual, C., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+machinery+and+regulation.">Autophagy: machinery and regulation.</a> Microbial Cell. 3 (12), 588-596 (2016).</li><li>Bodemann, B. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RalB+and+the+exocyst+mediate+the+cellular+starvation+response+by+direct+activation+of+autophagosome+assembly.">RalB and the exocyst mediate the cellular starvation response by direct activation of autophagosome assembly.</a> Cell. 144 (2), 253-267 (2011).</li><li>Bjorkoy, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p62/SQSTM1+forms+protein+aggregates+degraded+by+autophagy+and+has+a+protective+effect+on+huntingtin-induced+cell+death.">p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death.</a> The Journal of Cell Biology. 171 (4), 603-614 (2005).</li><li>Kabeya, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LC3,+a+mammalian+homologue+of+yeast+Apg8p,+is+localized+in+autophagosome+membranes+after+processing.">LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.</a> The EMBO Journal. 19 (21), 5720-5728 (2000).</li><li>Kabeya, Y., Mizushima, N., Yamamoto, A., Oshitani-Okamoto, S., Ohsumi, Y., Yoshimori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LC3,+GABARAP+and+GATE16+localize+to+autophagosomal+membrane+depending+on+form-II+formation.">LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation.</a> Journal of Cell Science. 117, 2805-2812 (2004).</li><li>Pankiv, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p62/SQSTM1+binds+directly+to+Atg8/LC3+to+facilitate+degradation+of+ubiquitinated+protein+aggregates+by+autophagy.">p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy.</a> The Journal of Biological Chemistry. 282 (33), 24131-24145 (2007).</li><li>Korolchuk, V. I., Rubinsztein, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+autophagy+by+lysosomal+positioning.">Regulation of autophagy by lysosomal positioning.</a> Autophagy. 7 (8), 927-928 (2011).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-based+quantitative+high-throughput+image+screening+identified+novel+autophagy+modulators.">A cell-based quantitative high-throughput image screening identified novel autophagy modulators.</a> Pharmacological Research. 110, 35-49 (2016).</li><li>Pampaloni, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Cellular+Spheroid-Based+Autophagy+Screen+Applying+Live+Fluorescence+Microscopy+Identifies+Nonactin+as+a+Strong+Inducer+of+Autophagosomal+Turnover.">A Novel Cellular Spheroid-Based Autophagy Screen Applying Live Fluorescence Microscopy Identifies Nonactin as a Strong Inducer of Autophagosomal Turnover.</a> SLAS Discovery. 22 (5), 558-570 (2017).</li><li>Deng, Y., Zhu, L., Cai, H., Wang, G., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagic+compound+database:+A+resource+connecting+autophagy-modulating+compounds,+their+potential+targets+and+relevant+diseases.">Autophagic compound database: A resource connecting autophagy-modulating compounds, their potential targets and relevant diseases.</a> Cell Proliferation. 51 (3), 12403 (2018).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beclin1+controls+the+levels+of+p53+by+regulating+the+deubiquitination+activity+of+USP10+and+USP13.">Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13.</a> Cell. 147 (1), 223-234 (2011).</li><li>Mauvezin, C., Neufeld, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bafilomycin+A1+disrupts+autophagic+flux+by+inhibiting+both+V-ATPase-dependent+acidification+and+Ca-P60A/SERCA-dependent+autophagosome-lysosome+fusion.">Bafilomycin A1 disrupts autophagic flux by inhibiting both V-ATPase-dependent acidification and Ca-P60A/SERCA-dependent autophagosome-lysosome fusion.</a> Autophagy. 11 (8), 1437-1438 (2015).</li><li>Yoshimori, T., Yamamoto, A., Moriyama, Y., Futai, M., Tashiro, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bafilomycin+A1,+a+specific+inhibitor+of+vacuolar-type+H(+)-ATPase,+inhibits+acidification+and+protein+degradation+in+lysosomes+of+cultured+cells.">Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.</a> Journal of Biological Chemistry. 266 (26), 17707-17712 (1991).</li><li>Gerer, K. F., Hoyer, S., Dorrie, J., Schaft, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+mRNA+as+Universal+Technology+Platform+to+Transfect+a+Variety+of+Primary+Cells+with+Antigens+and+Functional+Proteins.">Electroporation of mRNA as Universal Technology Platform to Transfect a Variety of Primary Cells with Antigens and Functional Proteins.</a> Methods in Molecular Biology. 1499, 165-178 (2017).</li><li>Prechtel, A. T., Turza, N. M., Theodoridis, A. A., Steinkasserer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD83+knockdown+in+monocyte-derived+dendritic+cells+by+small+interfering+RNA+leads+to+a+diminished+T+cell+stimulation.">CD83 knockdown in monocyte-derived dendritic cells by small interfering RNA leads to a diminished T cell stimulation.</a> The Journal of Immunology. 178 (9), 5454-5464 (2007).</li><li>Pfeiffer, I. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukoreduction+system+chambers+are+an+efficient,+valid,+and+economic+source+of+functional+monocyte-derived+dendritic+cells+and+lymphocytes.">Leukoreduction system chambers are an efficient, valid, and economic source of functional monocyte-derived dendritic cells and lymphocytes.</a> Immunobiology. 218 (11), 1392-1401 (2013).</li><li>Villadangos, J. A., Heath, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle,+migration+and+antigen+presenting+functions+of+spleen+and+lymph+node+dendritic+cells:+limitations+of+the+Langerhans+cells+paradigm.">Life cycle, migration and antigen presenting functions of spleen and lymph node dendritic cells: limitations of the Langerhans cells paradigm.</a> Seminars in Immunology. 17 (4), 262-272 (2005).</li><li>Lechmann, M., Berchtold, S., Hauber, J., Steinkasserer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD83+on+dendritic+cells:+more+than+just+a+marker+for+maturation.">CD83 on dendritic cells: more than just a marker for maturation.</a> Trends in Immunology. 23 (6), 273-275 (2002).</li><li>Yang, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+and+interpretation+of+current+autophagy+inhibitors+and+activators.">Application and interpretation of current autophagy inhibitors and activators.</a> Acta Pharmacologica Sinica. 34 (5), 625-635 (2013).</li><li>Redmann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+autophagy+with+bafilomycin+and+chloroquine+decreases+mitochondrial+quality+and+bioenergetic+function+in+primary+neurons.">Inhibition of autophagy with bafilomycin and chloroquine decreases mitochondrial quality and bioenergetic function in primary neurons.</a> Redox Biology. 11, 73-81 (2017).</li><li>Yan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bafilomycin+A1+induces+caspase-independent+cell+death+in+hepatocellular+carcinoma+cells+via+targeting+of+autophagy+and+MAPK+pathways.">Bafilomycin A1 induces caspase-independent cell death in hepatocellular carcinoma cells via targeting of autophagy and MAPK pathways.</a> Scientific Reports. 6, 37052 (2016).</li><li>Hale, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+modulators+of+autophagic+flux+in+an+image-based+high+content+siRNA+screen.">Identification of modulators of autophagic flux in an image-based high content siRNA screen.</a> Autophagy. 12 (4), 713-726 (2016).</li><li>Lipinski, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+siRNA+screen+reveals+multiple+mTORC1+independent+signaling+pathways+regulating+autophagy+under+normal+nutritional+conditions.">A genome-wide siRNA screen reveals multiple mTORC1 independent signaling pathways regulating autophagy under normal nutritional conditions.</a> Developmental Cell. 18 (6), 1041-1052 (2010).</li><li>Orvedahl, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+genome-wide+siRNA+screen+identifies+selective+autophagy+factors.">Image-based genome-wide siRNA screen identifies selective autophagy factors.</a> Nature. 480 (7375), 113-117 (2011).</li><li>Staskiewicz, L., Thorburn, J., Morgan, M. J., Thorburn, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibiting+autophagy+by+shRNA+knockdown:+cautions+and+recommendations.">Inhibiting autophagy by shRNA knockdown: cautions and recommendations.</a> Autophagy. 9 (10), 1449-1450 (2013).</li><li>Lee, I. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atg7+modulates+p53+activity+to+regulate+cell+cycle+and+survival+during+metabolic+stress.">Atg7 modulates p53 activity to regulate cell cycle and survival during metabolic stress.</a> Science. 336 (6078), 225-228 (2012).</li><li>Fremont, S., Gerard, A., Galloux, M., Janvier, K., Karess, R. E., Berlioz-Torrent, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beclin-1+is+required+for+chromosome+congression+and+proper+outer+kinetochore+assembly.">Beclin-1 is required for chromosome congression and proper outer kinetochore assembly.</a> EMBO Reports. 14 (4), 364-372 (2013).</li><li>Bestebroer, J., V'kovski, P., Mauthe, M., Reggiori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hidden+behind+autophagy:+the+unconventional+roles+of+ATG+proteins.">Hidden behind autophagy: the unconventional roles of ATG proteins.</a> Traffic. 14 (10), 1029-1041 (2013).</li><li>Galluzzi, L., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+and+divergent+functions+of+Beclin+1+and+Beclin+2.">Common and divergent functions of Beclin 1 and Beclin 2.</a> Cell Research. 23 (12), 1341-1342 (2013).</li></ol>

The authors have nothing to disclose.

The scope of the present protocol includes (i) the handling of human monocyte-derived iDCs as well as mDCs, (ii) their infection with HSV-1, (iii) their treatment with compounds known to inhibit autophagy, and (iv) their electroporation with siRNA using two different technical setups. Using the present protocol, autophagic flux can either be blocked in HSV-1-infected iDCs or induced in HSV-1-infected mDCs.
Since DCs, and especially iDCs, are very vulnerable cells, working with these cells involves rather delicate steps. For DC generation, we recommend to use freshly isolated PBMCs, and to avoid their cryopreservation, in order to obtain higher cell yields. Furthermore, when handling iDCs during experiments, including their subsequent cultivation, prevent harsh or prolonged temperature alterations. Otherwise, iDCs might undergo phenotypic changes and thus it is necessary to verify their immature phenotype by flow cytometry. Note, in contrast to their mature counterparts, iDCs lack distinct markers, such as CD80, CD83, and CD8628,29. The infection of iDCs and mDCs with HSV-1 is a well-established method2,3,4,5,6,10. We and others showed that DCs are highly susceptible for HSV-1 infection, when an MOI of 1 or 2 has been used (Figure 2). In our hands, keeping the volume of the infection medium at low levels (1-3 x 106 cells in 250-350 &#181;L) will lead to better infection efficiencies.
A classical approach to interfere with a given distinct cellular pathway is the usage of specific compounds. A variety of different modulators of autophagy, i.e. activators as well as inhibitors, are currently available30. Regarding HSV-1-induced autophagy in DCs, Turan et al., (2019) recently showed the inhibitory effects of spautin-1 and bafilomycin-A1 (BA1) on autophagic turnover in iDCs10. This technique for autophagy inhibition is suitable for the combination with a subsequent HSV-1 infection, since neither the infection rate nor the maturation status of DCs (especially iDCs) is impaired. In future applications, this inhibitor-based approach could be applied also in combination with other infectious agents, stress conditions, such as starvation, as well as for different cell types. However, when using inhibitors, limitations arise in determining the suitable concentration for efficient autophagy inhibition, without severely affecting cell viability. The major limitation when using inhibitors is, however, the occurrence of potential off-target or adverse effects, which could lead to misleading results31,32.
The second approach to interfere with autophagy, covered in the present protocol, is the specific knockdown using siRNA33,34,35. On the one hand, we used the electroporation apparatus I to specifically ablate the expression of FIP200, thereby inhibiting HSV-1-induced autophagic turnover in iDCs. On the other hand, we silenced two different KIF proteins (i.e., KIF1B and KIF2A), using the electroporation apparatus II, to facilitate autophagic flux in HSV-1-infected mDCs. Both electroporation protocols resulted in an almost complete ablation of FIP200 in iDCs, and KIF1B/KIF2A in mDCs, which was verified via Western blot analyses (Figure 4A, Figure 5A). In contrast to the electroporation apparatus I, which does not affect the viability of DCs, electroporation of mDCs using the electroporation apparatus II results in slightly higher rates of dead cells (Figure 4B, Figure 5B). Hence, in future applications, the electroporation apparatus I should be preferentially used for both, iDCs and mDCs. Remarkably, both siRNA-based techniques, to modulate autophagic flux, are compatible with subsequent HSV-1 infection of either iDCs or mDCs. Furthermore, neither the immature phenotype of iDCs nor the mature phenotype of mDCs is altered post electroporation.
Electroporation of iDCs using FIP200-specific siRNA is an efficient and highly specific method for gene knockdown as well as inhibition of autophagic flux upon HSV-1 infection. In addition to the specific silencing of FIP200, this protocol can be adapted to silence other autophagic components, participating at different steps during the autophagic cascade. However, identifying the appropriate target for efficient siRNA-mediated inhibition of autophagy includes several aspects of concern. Firstly, knockdown efficiency of autophagy-related genes (ATG) does not necessarily positively correlate with efficient inhibition of autophagy and is highly dependent on the specific ATG protein that is silenced36. Secondly, distinct ATG proteins are additionally involved in pathways distinct from autophagy, thus their ablation could also lead to adverse side effects37,38,39. Thirdly, different ATGs may have redundant functions, thus knockdown of one component may not be sufficient to inhibit autophagy (e.g., beclin-1 and beclin-2)40.
In addition, the electroporation apparatus I -based electroporation protocol of DCs is also suitable for mRNAs, and could be used for a variety of additional primary cell types, such as PBMCs25. This system thus provides a general strategy to deliver distinct RNA species into different primary cell types. In conclusion, we present two protocols to inhibit autophagic flux, by either using an inhibitor- or siRNA-based approach combined with subsequent HSV-1 infection of iDCs. Furthermore, we describe an siRNA electroporation approach to induce autophagic flux in mDCs upon HSV-1 infection.

The generation of human monocyte-derived dendritic cells (DCs) constitutes an appropriate in vitro model to study the functions and biology of this important immune cell type. Isolation as well as differentiation of monocytes into DCs has been well established in recent years1,2. The infection of DCs with the &#945;-herpesvirus herpes simplex virus type-1 (HSV-1) serves as a model system to study HSV-1-mediated modulations of DC biology2,3,4,5,6. This is particularly important to elucidate how herpesviruses dampen or inhibit potent antiviral immune responses, to establish latency in immune-privileged niches inside the host7,8. In this respect, herpesviruses are very successful pathogens that are wide-spread throughout the population reaching a sero-prevalence of up to 90&#160;% according to the geographic region9. To understand and possibly prevent this, more insights into the HSV-1-mediated modulations of the host&#8217;s immune system, and especially of immune cells such as DCs, are required.
A completely new observation regarding the interplay of DCs with HSV-1 was recently published by Turan et al.10. The authors demonstrated that the accomplishment of HSV-1 replication is strictly dependent on the maturation status of DCs. In iDCs, complete replication of HSV-1 is facilitated by autophagy-dependent mechanisms. While HSV1 induces autophagy in both, iDCs and mDCs, autophagic flux is observed only in iDCs. This in turn facilitates nuclear egress of viral capsids via autophagic degradation of nuclear lamins in iDCs. To gain mechanistic insights into this HSV-1-induced degradation pathway in iDCs versus mDCs, new and efficient strategies are critical to investigate autophagic flux.
Macroautophagy (autophagy) is a well-conserved multistep process targeting intracellular proteins or whole organelles for lysosomal digestion11. Simplistically, autophagy can be divided into the (i) initiation, (ii) membrane nucleation, (iii) vesicle expansion, and (iv) autophagosome-lysosome fusion phase12. During initiation (i), components such as the activated ULK1/2 kinase complex, containing the focal adhesion kinase family interacting protein of 200 kD (FIP200), are critical to activate the beclin-1-Vps34-AMBRA1 complex. Subsequently, membrane nucleation (ii) initiates phagophore formation13, which engulfs cytoplasmic cargos that are marked by molecules such as p6214. During vesicle expansion and autophagophore maturation (iii) microtubule-associated protein light chain 3 (LC3)-I is converted into its lipidated form LC3-II that is inserted into the autophagosomal membrane. Thus, LC3-I to -II conversion rates are an indicator for autophagy induction by mirroring the formation of mature autophagosomes15,16. Upon autophagosome-lysosome fusion (iv), not only the autophagic cargo but also associated p62 and LC3-II proteins undergo degradation (e.g., by hydrolysis). Thus, loss of p62 and LC3-II serve as markers for autophagic flux17. The fusion of autophagosomes with lysosomes, and thus following autophagic turnover, is highly dependent on the intracellular lysosomal localization. This is, among others, regulated by the kinesin family members KIF1B and KIF2A, which were shown to negatively affect autophagosome-lysosome fusion18. Interestingly, protein expression of KIF1B and KIF2A is induced upon DC maturation and is thereby responsible for the inefficient autophagic flux in HSV-1-infected mDCs, which hampers complete HSV-1 replication10.
Experimental attempts to modulate autophagy include the usage of compounds known to induce or inhibit this particular pathway19,20,21. In this study, we describe two inhibitor-based strategies to block autophagic turnover in HSV-1-infected iDCs. The first compound used in our experiments is specific and potent autophagy inhibitor-1 (spautin-1), which was described to promote beclin-1-Vps34-AMBRA1 complex degradation during the initiation phase of autophagy22. The second compound used in the present study is bafilomycin-A1 (BA1), a V-ATPase inhibitor that blocks the late autophagic events (i.e., autophagosome-lysosome fusion as well as autolysosome acidification)23,24. The usage of either of these two inhibitors prior to iDC infection with HSV-1 potently inhibits autophagy, but does not disturb efficient viral gene expression. Thus, this inhibitor-based strategy prior to HSV-1 infection offers a powerful tool to inhibit HSV-1-induced autophagic flux that can easily be expanded for a plethora of different cell types and viruses, which also potentially induce autophagy.
To overcome a major downside of an inhibitor-based approach (i.e., unspecific off-target effects), we developed an siRNA-based method to block autophagic flux in (HSV-1-infected) iDCs. The technique of siRNA electroporation represents a powerful alternative strategy, via selective ablation of the expression of distinct proteins (i.e., autophagic components). In our experiments iDCs were electroporated with FIP200-specific siRNA using the electroporation apparatus I (see Table of Materials) and a modified protocol described by Gerer et al. (2017) and Prechtel et al. (2007), to inhibit autophagy during the initiation phase25,26. This technique allowed us to specifically knockdown FIP200 expression in iDCs, without interfering with cell viability and their immature phenotype two days post electroporation. Noteworthy, HSV-1 infection was established in these electroporated iDCs mirrored by efficient viral protein expression. This siRNA-based technique offers a unique benefit (i.e., that a variety of different autophagic components, even in combination), can be specifically targeted for the ablation of their expression.
In this study, we further describe an siRNA-based method to induce autophagic flux also in HSV-1-infected mDCs. In this case, iDCs were electroporated with siRNA targeted against KIF1B and KIF2A prior to DC maturation using the electroporation apparatus II (see Table of Materials). Since both proteins are upregulated during DC maturation and known to negatively regulate fusion of autophagosomes with lysosomes10,18, their knockdown strongly induced autophagic flux in mDCs upon HSV-1 infection. Thus, the siRNA-based technique enabled us to specifically induce autophagic turnover via interfering with KIF protein expression in mDCs, and could thereby mimic their expression levels in iDCs.
In summary, we present two distinct methods to inhibit autophagic flux in HSV-1-infected iDCs. While the first inhibitor-based approach constitutes an easy, cheap and fast way to interfere with autophagic degradation, the second siRNA-based technique is more specific and a very suitable method to support and verify the results of inhibitor-based experiments. In addition, we describe a method to induce autophagic flux also in HSV-1-infected mDCs, via the siRNA-mediated knockdown of two KIF proteins.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4D-Nucleofector Core Unit (electroporation apparatus II)</td>
			<td>Lonza (Basel, Switzerland)</td>
			<td>AAF-1002B</td>
			<td></td>
		</tr>
		<tr>
			<td>AB-Serum</td>
			<td>Sigma Aldrich Chemie GmbH (Steinheim, Germany)</td>
			<td>H4522</td>
			<td>Dendritic cell cultivation</td>
		</tr>
		<tr>
			<td>ACD-A</td>
			<td>Sigma-Aldrich Chemie GmbH (Steinheim, Germany)</td>
			<td>9007281</td>
			<td></td>
		</tr>
		<tr>
			<td>Amaxa P3 Primary Cell 4D-Nucleofector X Kit L (electroporation kit apparatus II)</td>
			<td>Lonza (Basel, Switzerland)</td>
			<td>V4XP-3024</td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham ECL Prime Western Blotting Detection Reagent</td>
			<td>GE Healthcare (Solingen, Germany)</td>
			<td>RPN2232</td>
			<td>Western Blot Detection</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma Aldrich Chemie GmbH (Steinheim, Germany)</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse-IgG (mouse, polyclonal, HRP)</td>
			<td>Cell Signaling (Leiden, Netherlands)</td>
			<td>7076</td>
			<td>Western Blot detection</td>
		</tr>
		<tr>
			<td>anti-rabbit-IgG (goat, polyclonal, HRP)</td>
			<td>Cell Signaling (Leiden, Netherlands)</td>
			<td>7074</td>
			<td>Western Blot detection</td>
		</tr>
		<tr>
			<td>Bafilomycin A1</td>
			<td>Sigma-Aldrich Chemie GmbH (Steinheim, Germany)</td>
			<td>tlrl-baf1</td>
			<td>inhibition of autophagy and lysosomal degradation</td>
		</tr>
		<tr>
			<td>BD FACS Canto II Flow Cytometer</td>
			<td>BD Biosciences (Heidelberg, Germany)</td>
			<td>338962</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase</td>
			<td>Sigma-Aldrich Chemie GmbH (Steinheim, Germany)</td>
			<td>E1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Blotting Chamber Fastblot B44</td>
			<td>Biometra (G&#246;ttingen, Germany)</td>
			<td>846-015-100</td>
			<td></td>
		</tr>
		<tr>
			<td>CCR7 (mouse, Pe-Cy7)</td>
			<td>BioLegend (Fell, Germany)</td>
			<td>557648</td>
			<td>Flow cytometry 
			Dilution: 1:100 
			Clone: G043H7</td>
		</tr>
		<tr>
			<td>CD11c (mouse, Pe-Cy5)</td>
			<td>BD Biosciences (Heidelberg, Germany)</td>
			<td>561692</td>
			<td>Flow cytometry 
			Dilution: 1:100 
			Clone: B-ly6</td>
		</tr>
		<tr>
			<td>CD14 (mouse, PE)</td>
			<td>BD Biosciences (Heidelberg, Germany)</td>
			<td>555398</td>
			<td>Flow cytometry 
			Dilution: 1:100 
			Clone: M5E2</td>
		</tr>
		<tr>
			<td>CD3 (mouse, FITC)</td>
			<td>BD Biosciences (Heidelberg, Germany)</td>
			<td>555332</td>
			<td>Flow cytometry 
			Dilution: 1:100 
			Clone: UCHT1</td>
		</tr>
		<tr>
			<td>CD80 (mouse, V450)</td>
			<td>BD Biosciences (Heidelberg, Germany)</td>
			<td>560442</td>
			<td>Flow cytometry 
			Dilution: 1:100 
			Clone: L307.4</td>
		</tr>
		<tr>
			<td>CD83 (mouse, APC)</td>
			<td>eBioscience Thermo Fisher Scientific (Langenselbold, Germany)</td>
			<td>17-0839-41</td>
			<td>Flow cytometry 
			Dilution: 1:200 
			Clone: HB15e</td>
		</tr>
		<tr>
			<td>CD86 (mouse, PE)</td>
			<td>BD Biosciences (Heidelberg, Germany)</td>
			<td>553692</td>
			<td>Flow cytometry 
			Dilution: 1:100</td>
		</tr>
		<tr>
			<td>EVOS FL Cell Imaging</td>
			<td>System AMG/Life Technologies (Carlsbad, USA)</td>
			<td>AMF4300</td>
			<td></td>
		</tr>
		<tr>
			<td>FIP200 (rabbit)</td>
			<td>Cell Signaling (Leiden, Netherlands)</td>
			<td>12436</td>
			<td>Western Blot detection 
			Dilution: 1:1000 
			Clone: D10D11</td>
		</tr>
		<tr>
			<td>GAPDH (mouse)</td>
			<td>Merck Millipore (Massachusetts, USA)</td>
			<td>AB2302</td>
			<td>Western Blot detection 
			Dilution: 1:5000 
			Clone: MAB374</td>
		</tr>
		<tr>
			<td>Gene Pulser II apparatus (electroporation apparatus I)</td>
			<td>BioRad Laboratories GmbH (M&#252;nchen, Germany)</td>
			<td>165-2112</td>
			<td></td>
		</tr>
		<tr>
			<td>GM-CSF (4x104 U/mL)</td>
			<td>Miltenyi Biotec (Bergisch Gladbach, Germany)</td>
			<td>130-093-868</td>
			<td></td>
		</tr>
		<tr>
			<td>HLA&#8208;DR (mouse, APC-Cy7)</td>
			<td>BioLegend (Fell, Germany)</td>
			<td>307618</td>
			<td>Flow cytometry 
			Dilution: 1:200 
			Clone: L243</td>
		</tr>
		<tr>
			<td>HSV-1/17+/CMV-EGFP/UL43</td>
			<td>BioVex</td>
			<td></td>
			<td>DC infection 
			&#160;</td>
		</tr>
		<tr>
			<td>ICP0 (mouse)</td>
			<td>Santa Cruz Biotechnology (St. Cruz; Dallas, Texas, USA)</td>
			<td>sc-53070</td>
			<td>Western Blot detection 
			Dilution: 1:1000 
			Clone: 11060</td>
		</tr>
		<tr>
			<td>ICP5 (mouse)</td>
			<td>Santa Cruz Biotechnology (St. Cruz; Dallas, Texas, USA)</td>
			<td>sc-56989</td>
			<td>Western Blot detection 
			Dilution: 1:1000 
			Clone: 3B6</td>
		</tr>
		<tr>
			<td>IL-1&#946; (0.1x106 U/mL)</td>
			<td>Cell Genix GmbH (Freiburg, Germany)</td>
			<td>1411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-4 (1x106 U/mL)</td>
			<td>Miltenyi Biotec (Bergisch Gladbach, Germany)</td>
			<td>130-093-924</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-6 (1x106 U/mL)</td>
			<td>Cell Genix GmbH (Freiburg, Germany)</td>
			<td>1404-050</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageQuant LAS 4000</td>
			<td>GE Healthcare (Solingen, Germany)</td>
			<td>28955810</td>
			<td></td>
		</tr>
		<tr>
			<td>KIF1B (mouse)</td>
			<td>Santa Cruz Biotechnology (St. Cruz; Dallas, Texas, USA)</td>
			<td>sc-376246</td>
			<td>Western Blot detection 
			Dilution: 1:1000 
			Clone: E-12</td>
		</tr>
		<tr>
			<td>KIF2A (mouse)</td>
			<td>Santa Cruz Biotechnology (St. Cruz; Dallas, Texas, USA)</td>
			<td>sc-271471</td>
			<td>Western Blot detection 
			Dilution: 1:1000 
			Clone: D-7</td>
		</tr>
		<tr>
			<td>LC3B (rabbit)</td>
			<td>Cell Signaling (Leiden, Netherlands)</td>
			<td>3868</td>
			<td>Western Blot detection 
			Dilution: 1:1000 
			Clone: D11</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Lonza (Basel, Switzerland)</td>
			<td>17-605E</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Violet dead cell stain kit</td>
			<td>Life Technologies (Carlsbad, CA, USA)</td>
			<td>L34964</td>
			<td>L/D staining in Flow cytometry</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Alere Technologies AS (Oslo, Norway)</td>
			<td>04-03-9391/01</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesiumchloride</td>
			<td>Carl Roth GmbH (Karlsruhe, Germany)</td>
			<td>A537.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Megafuge 2.0 RS</td>
			<td>Heraeus (Hanau, Germany)</td>
			<td>75015505</td>
			<td></td>
		</tr>
		<tr>
			<td>N, N, N&#39;, N&#39;-Tetramethylethylendiamine (TEMED)</td>
			<td>Sigma-Aldrich Chemie GmbH (Steinheim, Germany)</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Brand (Wertheim, Germany)</td>
			<td>717805</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Cell culture flasks (175.0 cm2)</td>
			<td>Thermo Scientific (Rockford, USA)</td>
			<td>159910</td>
			<td></td>
		</tr>
		<tr>
			<td>p62 (rabbit)</td>
			<td>Cell Signaling (Leiden, Netherlands)</td>
			<td>88588</td>
			<td>Western Blot detection 
			Dilution: 1:1000 
			Clone: D5L7G</td>
		</tr>
		<tr>
			<td>PageRuler prestained protein ladder</td>
			<td>Thermo Fisher Scientific (Langenselbold, Germany)</td>
			<td>26616</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 16 %</td>
			<td>Alfa Aesar, Haverhill, USA</td>
			<td>43368.9M</td>
			<td></td>
		</tr>
		<tr>
			<td>PerfectSpin 24 Plus</td>
			<td>Peqlab (Erlangen, Germany)</td>
			<td>C2500-R-PL</td>
			<td></td>
		</tr>
		<tr>
			<td>PGE2 (1 mg/mL)</td>
			<td>Pfizer (Berlin, Germany)</td>
			<td>BE130681</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Lonza (Basel, Switzerland)</td>
			<td>17-512F</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein gel system MiniProtean II</td>
			<td>Bio-Rad Laboratories GmbH (M&#252;nchen, Germany)</td>
			<td>1652960</td>
			<td></td>
		</tr>
		<tr>
			<td>RestoreTM Western Blot Stripping Buffer</td>
			<td>Thermo Scientific, Rockford, USA</td>
			<td>21059</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking Platform wt 15</td>
			<td>Biometra (G&#246;ttingen, Germany)</td>
			<td>042-590</td>
			<td></td>
		</tr>
		<tr>
			<td>RotiBlock</td>
			<td>Carl Roth GmbH (Karlsruhe, Germany)</td>
			<td>A151.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Roti-Load 1 (4x)</td>
			<td>Carl Roth GmbH (Karlsruhe, Germany)</td>
			<td>K929.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotiphorese Gel 30 (37.5:1)</td>
			<td>Carl Roth GmbH (Karlsruhe, Germany)</td>
			<td>3029.1</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Lonza (Basel, Switzerland)</td>
			<td>12-167F</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl Sulfate (SDS)</td>
			<td>Carl Roth GmbH (Karlsruhe, Germany)</td>
			<td>2326.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer comfort</td>
			<td>Eppendorf (Hamburg, Germany)</td>
			<td>5355 000.011</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF-&#945; (10 &#956;g/mL)</td>
			<td>Peprotech (Hamburg, Germany)</td>
			<td>300-01A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Carl Roth GmbH (Karlsruhe, Germany)</td>
			<td>4855.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue solution (0.4 %)</td>
			<td>Sigma-Aldrich Chemie GmbH (Steinheim, Germany)</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Carl Roth GmbH (Karlsruhe, Germany)</td>
			<td>9127.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman 0.2 &#956;m nitrocellulose membrane</td>
			<td>GE Healthcare (Solingen, Germany)</td>
			<td>10600001</td>
			<td></td>
		</tr>
		<tr>
			<td>WhatmanTM Chromatography Paper 3 mm Chr</td>
			<td>Fisher Scientific GmbH (Schwerte, Germany)</td>
			<td>3030917</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 manuscript, we describe methods to interfere with HSV-1-induced autophagy in dendritic cells. This includes the generation of human monocyte-derived iDCs and mDCs, which were phenotypically analyzed by flow cytometry (Figure 1). On day 4 post adherence, DCs show an immature phenotype characterized by weak expression of CD80, CCR7, and CD83 as well as high CD11c and intermediate MHCII expression. Since CD3 and CD14 signals are missing, T cell and monocyte contaminations can be excluded. On day 6 post adherence (i.e., day 2 post induction of maturation), DCs show a mature phenotype reflected by a significant increase in CD80, CCR7, CD83, and MHC-II surface expression. Infection with an eGFP-expressing HSV-1 strain (Figure 2) results in an almost complete infection of either iDCs (Figure 2A upper panel) or mDCs (Figure 2A lower panel, Figure 2B), based on strong GFP signals analyzed by fluorescence microscopy as well as flow cytometry.
As demonstrated in our recent report, HSV-1 induces autophagy both in iDCs and mDCs, however, autophagic turnover occurs in iDCs only10. In a first approach, we treated iDCs and mDCs with spautin-1 (Figure 3A) - to block autophagy initiation - or bafilomycin-A1 (BA1; Figure 3B) - to inhibit final autophagosome-lysosome fusion. Upon HSV-1 infection of iDCs in the absence of spautin-1 and BA1, autophagic flux is mirrored by the decline of p62 and LC3B expression, respectively. In contrast, HSV-1 infection of mDCs in the absence of spautin-1 does not affect p62 expression, while spautin-1 and BA1 treatment induce an accumulation of LC3B-II. This reflects the induction of autophagy but a failure of autophagic turnover in mDCs. In iDCs, spautin-1 pre-treatment strongly restores autophagic degradation of p62 upon HSV-1 infection, due to the inhibition of autophagy during the initiation phase. Upon pre-treatment with BA1, mock- and HSV-1-infected iDCs show a strong accumulation of LC3B-II protein levels, indicating successful inhibition of autophagic turnover via blocking late autophagosome-lysosome fusion. Consistent with this, spautin-1 and BA1 pre-treatment of mDCs also results in stable p62 and increased LC3B-II protein levels, respectively.
In a second method to impair autophagic flux, siRNA electroporation targeting FIP200 is examined regarding its capacity to block autophagic flux in HSV-1-infected iDCs. As shown in Figure 4A, strongly reduced FIP200 protein levels were detected in iDCs 48 h post electroporation, compared to control siRNA. At this time point, iDCs do not show any signs of cell death (Figure 4B) and maintain their immature phenotype (Figure 4C). Infection of FIP200-silenced iDCs with HSV-1 reveals a strong decrease in autophagic flux compared to their control siRNA-treated counterparts (Figure 4D). This is accompanied by increased protein levels of LC3B as well as p62 when FIP200 is silenced in HSV-1-infected iDCs.
In a reverse attempt, we studied whether siRNA-mediated ablation of KIF1B and KIF2A protein expression enables autophagosomal-lysosomal turnover also in HSV-1-infected mDCs. Thus, iDCs were electroporated using specific siRNAs targeting either one or both of these proteins, and cells were subsequently matured (Figure 5). 2 days post electroporation, mDCs show a strong reduction in KIF1B and/or KIF2A protein expression, when specific siRNAs were used (Figure 5A). This method also did neither lead to prominent cell death (Figure 5B) nor to changes in their phenotypic maturation status (Figure 5C). Supporting the importance of KIF1B and KIF2A during autophagosomal-lysosomal degradation, their depletion prior to HSV-1 infection facilitates an increased autophagic flux in mDCs. This is reflected by decreased residual p62 protein levels, in contrast to the respective control condition (Figure 5D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60190/60190fig01.jpg" /> 
Figure 1: Phenotypic characterization of human monocyte-derived iDCs and mDCs using flow cytometry.&#160;DCs were generated and stained with specific antibodies to verify their purity: (A) CD3 to exclude T cell contaminations, (B) CD14 to exclude contamination with monocytes, and (C) CD11c as a marker for DCs. To assess their phenotypic maturation status the following antibodies were used: (D) CD80, (E) CCR7, (F) CD83, and (G) MHCII. These molecules are highly expressed on mDCs and thus allow the discrimination between the immature and mature DC phenotype. Data were analyzed using FCS express 5.0. <a href="https://www.jove.com/files/ftp_upload/60190/60190fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60190/60190fig02.jpg" /> 
Figure 2: Microscopic as well as flow cytometric analyses of HSV-1-infected iDCs and mDCs.&#160;iDCs and mDCs were infected with an HSV-1 strain expressing EGFP (HSV-1 EGFP), to allow quantification of the infection rate based on the GFP signal. (A) Microscopic analyses of GFP-positive HSV-1-infected iDCs and mDCs infected at an MOI of 2, compared to their uninfected counterparts, at 24 hpi. To visualize infected cells, GFP fluorescence was monitored. Scale bar represents 400 &#181;m. (B) Flow cytometric measurement of mock- or HSV-1-infected mDCs during infection kinetics. Upper panels (black lined histograms) show mock condition, lower panels (black filled histograms) show HSV-1-infected cells after the indicated time points post infection. Data were analyzed using FCS express 5.0. <a href="https://www.jove.com/files/ftp_upload/60190/60190fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60190/60190fig03.jpg" /> 
Figure 3: Spautin-1 and b afilomycin-A1 modulate the autophagic flux in HSV-1-infected iDCs.&#160;iDCs and mDCs were treated with (A) spautin-1 or (B) bafilomycin-A1 (BA1) for 1 h prior to infection. Cells were subsequently mock- or HSV-1-infected (HSV1-RFPVP26) using an MOI of 2. After 16-18 h, DCs were harvested and protein lysates were subjected to Western blotting to determine expression of p62 or LC3B-I/-II as autophagic markers, ICP0 as infection control, and GAPDH as loading control. LC3B-I and LC3B-II protein levels were quantified and normalized to the reference protein GAPDH using Bio1D (optical density). The ratio of normalized LC3B-II to normalized LC3B-I signals is shown. This figure has been modified and adapted from &#169;2019 Turan et al. originally published in JCB. https://doi.org/10.1083/jcb.20180115110. <a href="https://www.jove.com/files/ftp_upload/60190/60190fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60190/60190fig04.jpg" /> 
Figure 4: Analysis of autophagic flux in HSV-1-infected iDCs upon FIP200-siRNA electroporation.&#160;iDCs were electroporated with control siRNA or FIP200-specific siRNA using the electroporation apparatus I. (A) DCs were analyzed regarding the efficiency of FIP200 knockdown 48&#160;h post electroporation, by performing Western blot analyses. (B) Cell viability as well as (C) maturation status was analyzed prior to electroporation (light blue histograms) and 48 h post (dark blue and grey histograms) electroporation by flow cytometry. Median values for three different donors are shown. After confirming efficient knockdown of FIP200 and the immature phenotype, cells were HSV-1-infected (HSV-1 EGFP) using an MOI of 2. Data were analyzed using FCS express 5.0. (D) At 20 h post infection, cells were subjected to Western blot analyses to determine expression of LC3B-I/-II and p62 as autophagic markers. ICP5 was detected as infection control, and GAPDH as loading control. Panels A and D have been modified and adapted from &#169;2019 Turan et al. originally published in JCB. https://doi.org/10.1083/jcb.20180115110. <a href="https://www.jove.com/files/ftp_upload/60190/60190fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60190/60190fig05.jpg" /> 
Figure 5: siRNA-mediated ablation of KIF1B and/or KIF2A modulates autophagic turnover in HSV-1-infected mDCs.&#160;iDCs were electroporated with KIF1B-specific and/or KIF2A-specific siRNA, as well as control siRNA, using the electroporation apparatus II. At 4 h post electroporation, maturation was induced via addition of a maturation cocktail. At 48 h post electroporation, DCs were analyzed regarding (A) the efficiency of KIF knockdown via Western blotting, (B) cell viability as well as their (C) phenotypic maturation status using flow cytometric analyses (two different donors are shown). &#34;w/o EP&#34;&#160;means without electroporation, but post induction of maturation; &#34;post control EP&#34;&#160;means post electroporation using control siRNA; &#34;post KIF1B, KIF2A, KIF1B/2A EP&#34;&#160;means post electroporation using KIF1B- and/or KIF2A-specific siRNA. After confirming efficient knockdown of KIF1B and/or KIF2A and the mature phenotype, cells were HSV-1-infected (HSV-1 EGFP) using an MOI of 2. Data were analyzed using FCS express 5.0. (D) Cells were subjected to Western blot analyses 20 h post infection, in order to assess expression of p62 as an autophagic marker. ICP5 was used as an infection control, and GAPDH as loading control. Figures A and D have been modified and adapted from &#169;2019 Turan et al. originally published in JCB. https://doi.org/10.1083/jcb.20180115110. <a href="https://www.jove.com/files/ftp_upload/60190/60190fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells at JoVE.com

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

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.

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas ...

sirna-electroporation-to-modulate-autophagy-herpes-simplex-virus-type

Research

JoVE Journal

Immunology and Infection

7.1K Views.  Universitätsklinikum Erlangen. 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.

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

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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.

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 ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

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 ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.