siRNA-Based Inhibition of Autophagy in Herpes Simplex Virus-Infected Immature Dendritic Cells

Protocol

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 "Friedrich-Alexander-Universität Erlangen-Nürnberg" (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)

1. Isolate human peripheral blood mononuclear cells (PBMCs) from leukoreduction system chambers (LRSCs) as previously described. Avoid cryopreservation of PBMCs and use them directly upon isolation to obtain higher DC yields.
2. Generate human DCs from PBMCs of different healthy donors in T175 cell culture flasks as previously described. Briefly, use 350-400 millions of PBMCs in 30 mL of DC medium (RPMI 1640 without L-glutamine, 1% (v/v) Human Serum Type AB (AB-serum), 100 U/mL penicillin, 100 mg/mL streptomycin, 0.4 mM L-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (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 granulocyte-macrophage colony-stimulating factor (GM-CSF) and 250 U/mL interleukin-4 (IL-4), and incubate for 3 days.
   1. 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.
   2. 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), interleukin-6 (IL-6, final concentration: 1000 U/mL), Interleukin-1 beta (IL-1β, final concentration: 200 U/mL), Tumor Necrosis Factor alpha (TNF-α, final concentration: 10 ng/mL), prostaglandin E2 (PGE2; final concentration: 1 μg/mL).
   3. 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.
3. 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.
   1. Gently resuspend (=wash) DCs in 5-10 mL of RPMI 1640 per cell culture flask. Combine respective DC suspensions in one tube.
   2. 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.

2. Flow cytometric analyses to monitor the phenotypic maturation status

1. 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)).
2. Resuspend the cells in 100 µL of antibody staining solution (FACS buffer) containing specific fluorochrome-labeled antibodies against defined surface molecules.
   1. Use the following antibodies to verify purity (CD3- fluorescein isothiocyanate [FITC], CD14- phycoerythrin [PE]) as well as maturation status of DCs (CD80-PacBlue [Pacific Blue]/-PE- cyanine5 [Cy5], CD11c-PE-Cy5, CCR7-PE-Cy7, CD83-APC, CD86-PE, MHCII-APC-Cy7).
   2. Prepare one unstained sample in 100 µL of FACS buffer as a control.
   3. Stain the cells on ice in the dark for 30 min.
3. Subsequently wash the cells two times in 1 mL of FACS buffer and centrifuge at 3390 x g for 1.5 min.
4. Finally, resuspend the cells in 200 µL of FACS buffer supplemented with 2% paraformaldehyde (PFA) and analyze the cells by flow cytometry. Fixed cells can be stored at 4° C in the dark up to 2 days.

3. 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).

1. 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).
2. 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 µL of OptiMEM without phenol red, adjusting a cell concentration of 6 x 10⁶/100 µL. 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.
3. Transfer either 75 pmol of FIP200-specific siRNA or 75 pmol of scrambled siRNA, as a control, into 4 mm electro cuvettes and add 100 µL (6x10⁶ cells) of the cell suspension. Directly pulse iDCs using the electroporation apparatus I, applying the following settings: 500 V for 1 ms.
   1. Prior to the experimental procedure, prepare siRNA suspensions according to the manufacturer's instruction, aliquot and store them at -20 °C. Thaw and keep them on ice when using these siRNAs for electroporation. Before electroporating the samples, perform a test pulse.
4. 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.
5. 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% ethylenediaminetetraacetic acid (EDTA) and transfer the solution in the respective tubes.
6. Subsequently, split 6 x 10⁶ iDCs per siRNA condition as described in the next steps.
   1. 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.
   2. 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 and perform Western blot analyses.
   3. 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 multiplicity of infection (MOI) of 2 or add MNT buffer as a mock control. Perform the infection as described.
   4. At 20 h post infection (hpi), harvest the cells using a cell scraper and prepare cell lysates for Western blot analyses.

Materials

Name	Company	Catalog Number	Comments
4D-Nucleofector Core Unit (electroporation apparatus II)	Lonza (Basel, Switzerland)	AAF-1002B
AB-Serum	Sigma Aldrich Chemie GmbH (Steinheim, Germany)	H4522	Dendritic cell cultivation
ACD-A	Sigma-Aldrich Chemie GmbH (Steinheim, Germany)	9007281
Amaxa P3 Primary Cell 4D-Nucleofector X Kit L (electroporation kit apparatus II)	Lonza (Basel, Switzerland)	V4XP-3024
Amersham ECL Prime Western Blotting Detection Reagent	GE Healthcare (Solingen, Germany)	RPN2232	Western Blot Detection
Ammonium persulfate (APS)	Sigma Aldrich Chemie GmbH (Steinheim, Germany)	A3678
anti-mouse-IgG (mouse, polyclonal, HRP)	Cell Signaling (Leiden, Netherlands)	7076	Western Blot detection
anti-rabbit-IgG (goat, polyclonal, HRP)	Cell Signaling (Leiden, Netherlands)	7074	Western Blot detection
BD FACS Canto II Flow Cytometer	BD Biosciences (Heidelberg, Germany)	338962
Blotting Chamber Fastblot B44	Biometra (Göttingen, Germany)	846-015-100
CCR7 (mouse, Pe-Cy7)	BioLegend (Fell, Germany)	557648	Flow cytometry Dilution: 1:100 Clone: G043H7
CD11c (mouse, Pe-Cy5)	BD Biosciences (Heidelberg, Germany)	561692	Flow cytometry Dilution: 1:100 Clone: B-ly6
CD14 (mouse, PE)	BD Biosciences (Heidelberg, Germany)	555398	Flow cytometry Dilution: 1:100 Clone: M5E2
CD3 (mouse, FITC)	BD Biosciences (Heidelberg, Germany)	555332	Flow cytometry Dilution: 1:100 Clone: UCHT1
CD80 (mouse, V450)	BD Biosciences (Heidelberg, Germany)	560442	Flow cytometry Dilution: 1:100 Clone: L307.4
CD83 (mouse, APC)	eBioscience Thermo Fisher Scientific (Langenselbold, Germany)	17-0839-41	Flow cytometry Dilution: 1:200 Clone: HB15e
CD86 (mouse, PE)	BD Biosciences (Heidelberg, Germany)	553692	Flow cytometry Dilution: 1:100
EVOS FL Cell Imaging	System AMG/Life Technologies (Carlsbad, USA)	AMF4300
FIP200 (rabbit)	Cell Signaling (Leiden, Netherlands)	12436	Western Blot detection Dilution: 1:1000 Clone: D10D11
Gene Pulser II apparatus (electroporation apparatus I)	BioRad Laboratories GmbH (München, Germany)	165-2112
GM-CSF (4x104 U/mL)	Miltenyi Biotec (Bergisch Gladbach, Germany)	130-093-868
HLA-DR (mouse, APC-Cy7)	BioLegend (Fell, Germany)	307618	Flow cytometry Dilution: 1:200 Clone: L243
HSV-1/17+/CMV-EGFP/UL43	BioVex		DC infection
IL-1β (0.1x106 U/mL)	Cell Genix GmbH (Freiburg, Germany)	1411-050
IL-4 (1x106 U/mL)	Miltenyi Biotec (Bergisch Gladbach, Germany)	130-093-924
IL-6 (1x106 U/mL)	Cell Genix GmbH (Freiburg, Germany)	1404-050
ImageQuant LAS 4000	GE Healthcare (Solingen, Germany)	28955810
L-glutamine	Lonza (Basel, Switzerland)	17-605E
LIVE/DEAD Fixable Violet dead cell stain kit	Life Technologies (Carlsbad, CA, USA)	L34964	L/D staining in Flow cytometry
Lymphoprep	Alere Technologies AS (Oslo, Norway)	04-03-9391/01
Magnesium chloride	Carl Roth GmbH (Karlsruhe, Germany)	A537.1
Megafuge 2.0 RS	Heraeus (Hanau, Germany)	75015505
Neubauer counting chamber	Brand (Wertheim, Germany)	717805
Nunc Cell culture flasks (175.0 cm2)	Thermo Scientific (Rockford, USA)	159910
Paraformaldehyde, 16 %	Alfa Aesar, Haverhill, USA	43368.9M
PerfectSpin 24 Plus	Peqlab (Erlangen, Germany)	C2500-R-PL
PGE2 (1 mg/mL)	Pfizer (Berlin, Germany)	BE130681
Phosphate buffered saline (PBS)	Lonza (Basel, Switzerland)	17-512F
RestoreTM Western Blot Stripping Buffer	Thermo Scientific, Rockford, USA	21059
Rocking Platform wt 15	Biometra (Göttingen, Germany)	042-590
RotiBlock	Carl Roth GmbH (Karlsruhe, Germany)	A151.4
Roti-Load 1 (4x)	Carl Roth GmbH (Karlsruhe, Germany)	K929.3
Rotiphorese Gel 30 (37.5:1)	Carl Roth GmbH (Karlsruhe, Germany)	3029.1
RPMI 1640	Lonza (Basel, Switzerland)	12-167F
Sodium dodecyl Sulfate (SDS)	Carl Roth GmbH (Karlsruhe, Germany)	2326.2
Thermomixer comfort	Eppendorf (Hamburg, Germany)	5355 000.011
TNF-α (10 μg/mL)	Peprotech (Hamburg, Germany)	300-01A
Tris	Carl Roth GmbH (Karlsruhe, Germany)	4855.3
Trypan blue solution (0.4 %)	Sigma-Aldrich Chemie GmbH (Steinheim, Germany)	T8154
Tween 20	Carl Roth GmbH (Karlsruhe, Germany)	9127.1
Whatman 0.2 μm nitrocellulose membrane	GE Healthcare (Solingen, Germany)	10600001
WhatmanTM Chromatography Paper 3 mm Chr	Fisher Scientific GmbH (Schwerte, Germany)	3030917