Our protocol describes methods to efficiently interfere with HSV-1 induced autophagic turnover in dendritic cells. And in particular, we compare an inhibitor-based with an siRNA-based strategy to block autophagy prior to infection. While the inhibitor-based interference with autophagy comprises potential and specific off-target effects, our developed siRNA-based strategy is more target specific.
Importantly, both presented techniques do not affect the maturation stages of disease. Demonstrating the procedure will be Petra Muhl-Zurbes, a technician, and Alexandra Duthorn, a grad student, from my laboratory. Begin by harvesting immature dendritic cells or iDCs by gently rinsing the loosely adherent cells from the bottom of the cell culture flask on day four post adherence.
Add maturation cocktail to the cells to generate mature dendritic cells or mDCs. Two days after induction of maturation, rinse the mDCs from the bottom of the cell culture flask. Repeat the rinse twice, then transfer the iDCs and mDCs to 50 milliliter tubes in their respective culture medium.
Harvest the cells via centrifugation at 300 times g for five minutes. Gently resuspend the cells in five to 10 milliliters of RPMI 1640 per cell culture flask and combine the respective DC suspensions in one tube. Then quantify the cells using a counting chamber making sure to avoid temperature alterations when handling the DCs.
Transfer two million iDCs or mDCs into a two milliliter tube, centrifuge them at 3, 390 times g for 1-1/2 minutes and discard the supernatant. Gently the resuspend the cells in infection medium. Inhibit the autophagosomal lysosomal degradation pathway by adding 10 micromolar spautin-1 or one micromolar bafilomycin A1 to the infection medium one hour prior to infection.
Add DMSO for the untreated control and incubate the cells on a heating block at 37 degrees Celsius with shaking at 300 RPM. For infection studies, inoculate the cells with HSV-1 virions at a multiplicity of infection of two. Add the respective volume of MNT buffer as a mock control and incubate the cells on a heating block at 37 degrees Celsius for one hour with shaking.
One hour after infection, collect the cells via centrifugation at 3, 390 times g for 1-1/2 minutes, then aspirate the inoculum gently and resuspend the cells in DC medium. Seed mock treated and HSV-1 infected cells at a final concentration of one million cells per milliliter into a six-well plate. At 16 to 24 hours post infection, harvest iDCs using a cell scraper or mDCs by rinsing, transfer the cells into a 1.5 milliliter safe-lock tube and collect them via centrifugation at 3, 390 times g for 1-1/2 minutes.
Repeat harvesting and centrifugation with the remaining cells in the wells, then wash the pellet once with one milliliter of PBS and vigorously resuspend the cells in lysis mix. Incubate the samples at 37 degrees Celsius for 10 minutes and then heat to 95 degrees Celsius for 10 minutes. Perform SDS page and western blot analysis to verify LC3B1 and LC3B2, P62, ICP0 and ICP5, and GAPDH protein levels.
On day 3.5 post adherence, transfer 12 million iDCs into a 50 milliliter tube, centrifuge them at 300 times g for five minutes, and then discard the supernatant. In parallel, perform flow cytometric analysis to monitor the maturation status. Gently wash the iDCs in five milliliters of Opti-MEM without phenol red and centrifuge them at 300 times g for five minutes.
Discard the supernatant and resuspend the cells in 200 microliters of Opti-MEM adjusting the cell concentration to six million cells per 100 microliters. Add 75 picomoles of either FIP200 specific or scrambled siRNA to four millimeter electrocuvettes and transfer 100 microliters of the cell suspension into the respective cuvette. Directly pulse the iDCs using an electroporation apparatus.
After electroporation, transfer the iDCs into six-well plates with fresh pre-warmed DC medium, seeding the cells at a final concentration of one million cells per milliliter and place them in the incubator. After 48 hours, examine the morphology of the electroporated iDCs microscopically, then harvest the cells with the cell scraper and transfer them into 15 milliliter tubes. Rinse the wells with one milliliter of PBS supplemented with 0.1%EDTA and transfer the solution to the respective tubes.
Then split the cells for each siRNA condition as described in the manuscript. Use 500, 000 cells to assess maturation status and cell viability, use one million cells for western blot analysis to verify FIP200 specific knockdown efficiency and use the remaining cells for HSV-1 infection experiments. The generated iDCs and mDCs were phenotypically analyzed by flow cytometry in order to exclude contamination with other cell types and to verify their maturation status.
The cells were stained with CD3 and CD14 antibodies to exclude T-cell and monocyte contamination respectively. The cells were stained for CD11c which serves as a highly expressed general marker for DCs. To assess maturation status, CD80, CCR7, CD83, and MHCII antibodies were used because these molecules are highly expressed on mature DCs.
Fluorescence microscopy and flow cytometry were used to determine infection with EGFP expressing HSV-1. Strong GFP signals indicate almost complete infection of iDCs and mDCs. Western blot analysis was used to determine if spautin-1 or bafilomycin A1 inhibit autophagic flux in HSV-1 infected cells.
In iDCs, autophagic flux is demonstrated by the decline of P62 and LC3B expression in the absence of spautin-1 and bafilomycin A1 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 induced an accumulation of LC3B-II which reflects the induction of autophagy but a failure of autophagic turnover in mDCs. Reducing FIP200 protein levels with siRNA electroporation also causes a strong decrease in autophagic flux in HSV-1 infected iDCs.
This siRNA-based electroporation protocol for inhibition of autophagy does not affect the cell viability, the image phenotype or the establishment of HSV-1 protein expression in iDCs. Our electroporation protocols offer the opportunity to deliver distinct DNA or RNA species into disease and other primary cell types. Subsequently, a variety of molecular, functional, and infection analyses can be performed.
The siRNA-based strategy for expression silencing in disease paves the way to explore the function of any protein of interest, also combined with subsequent functional assays.
