Methods for the Measurement of Early Events in Toxoplasma gondii Immunity in Mouse Cells

Abstract

Critical steps in resistance of mice against Toxoplasma gondii occur in the first 2 or 3 h after the pathogen has entered a cell that has been exposed to interferon γ (IFNγ). The newly formed parasitophorous vacuole is attacked by the IFNγ-inducible IRG proteins and disrupted, resulting in death of the parasite and necrotic death of the cell. Here we describe some techniques that we have used to describe and quantify these events in different combinations of the host and the parasite.

Appendices

Appendix 1: Generating Mammalian Cell Lines Expressing an HMGB1-GFP Necrosis Reporter (Joana Loureiro)

1.1 Generation of the Mouse HMGB1-GFP Construct

A murine HMGB1 cDNA (mHMGB1) with an in-frame C-terminal eGFP moiety (mHMGB1-GFP) was shown to be functionally similar to untagged, endogenous HMGB1 [24]. Murine messenger RNA (mRNA) obtained using the Qiagen RNeasy Kit (Cat No. 74104) from C57BL/6J MEFs induced for 24 h with murine IFNγ. RNA was DNase-treated (Thermo Fisher Scientific TURBO™ DNase, Cat No. AM2238) prior to cDNA synthesis (Invitrogen SuperScript III First-Strand Synthesis System, Cat No. 18080051). The 647 bp coding sequence of HMGB1 (Gene ID: 15289) was amplified from this cDNA (Herculase II Fusion DNA Polymerase, Agilent Technologies (Cat No. 600675) with primers JL1 and JL2 (see Table 3, below). After purification from an agarose gel (High Pure PCR Product Purification kit, Roche Cat. No. 11732668001), the amplicon was digested with EcoRI and SacII and cloned into EcoRI/SacII-digested Clontech’s pEGFP-N3 plasmid (now discontinued), ligated using Gibson Assembly^® Master Mix (New England Biolabs, Cat. No. E2611S) and transformed into One Shot^® Mach1™-T1R Chemically Competent E. coli. Positive colonies were identified by colony PCR and the sequence of the fusion gene in the pEGFP-N3 expression vector confirmed using Primer S1.

A functional HMGB1-GFP fusion protein should display a mostly nuclear localization in living mammalian cells, consistent with the nucleus-cytoplasm shuttling dynamics of endogenous untagged HMGB1 [22]. Indeed, cells with green-fluorescent nuclei were readily visualized and a protein of the expected size (~50–60 kDa)—GFP protein (238 aa) ~27 kDa; mHMGB1 (215 aa) ~20–30 kDa—was detected by western blot after transfection of pEGFP-N3 expressing the mHMGB1-GFP fusion construct into HEK293T cells (ATCC^® CRL-3216™) (data not shown).

1.2 Generating the Retroviral mHGMB1-GFP Expression Construct

Because transfection efficiencies are often low in primary cells, we made a high-titer helper-free retroviral vector based on the pM6pBLAST system [25]: we used primers JL3 and JL4 (Table 3) to subclone the pEGFP-N3 mHMGB1-GFP cassette into pM6pBLAST. The ~1400 bp was digested with NcoI and NotI and cloned into Nco1/Not1 pM6pBLAST. A clone confirmed to contain the correct sequence (primers S2 and S3) showed the expected protein behavior both by microscopy and western blot following transfection of the viral plasmid DNA into HEK 293Tcells (data not shown).

Table 3 Primers used in generation of murine HMGB1-GFP constructs

The pM6pBLAST-based viral plasmid was used to produce high-titer retroviruses expressing the mHMGB1-GFP gene through cotransfection with a packaging plasmid into HEK 293Tcells, and the retroviral supernatants of HEK 293T cultures were used to transduce mouse or human fibroblasts. Both procedures are described in detail below.

1.3 Generation of Mammalian Cell Lines Expressing the HMGB1-GFP Reporter

In our experience, retrovirus-mediated transduction of mouse and human fibroblasts is desirable for several reasons. It is highly efficient: we typically obtain >70% of cells displaying green nuclei within 1 day of HMGB1-GFP-retrovirus-mediated transduction. Moreover, retroviral transduction results in stable integration of the HMGB1-GFP cassette into the genome, leading to long-term expression. Additionally, the Blasticidin-S resistance gene present in pM6pBLAST allows enrichment for HMGB1-GFP expression in cells in culture by means of antibiotic (blasticidin) selection.

HEK293T cells used for virus packaging must be of low passage and must be very carefully maintained such that they never reach confluence; the viruses must be packaged with a viral envelope appropriately pseudotyped to ensure mouse or human cell tropism (seeNote 1, below); the efficiency of virus transduction goes down exponentially with freeze-thawing of the virus-containing HEK293T cell culture supernatant, and therefore it is preferable to coordinate packaging and transduction so as to use freshly packaged virus.

1.3.1 Protocol for HMGB1-GFP Virus Production in HEK293T Cells

1.3.1.1 Material

• 1. DMEM supplemented with 10% heat-inactivated FCS, 1× NEAA, 1× penicillin–streptomycin, 1 mM sodium pyruvate, and 2 mM l-glutamine.

• 2. Cell culture incubator maintained at 37 °C with 10% CO₂.

• 3. Sterile Flat bottom 6-well plates (Tissue culture-treated).

• 4. Pipette filter tips (20, 200, and 1000 μl).

• 5. Serological Pipettes (5 and 10 ml).

• 6. 1.5 ml sterile microcentrifuge tubes.

• 7. ScreenFect^®A plasmid transfection reagent and buffer (Incella Cat No. S-3001).

• 8. Retroviral DNA plasmid expressing murine HMGB1-GFP (fusion construct in pM6p.BLAST).

• 9. Packaging plasmid: pCL-Eco (mouse-pseudotyped envelope) plasmid or pCl-10A1 (human-pseudotyped envelope) [42].

• 10. 15 ml Falcon tubes.

• 11. 5 ml Plastic syringes.

• 12. Syringe Filters (Acrodisc^® 25 mm Syringe filters with 0.45 μm Supor^® Membrane).

1.3.1.2 Methods

Day −1: (Day before transfection) Plate cells at 750,000 cells/well in 6-well plates.

Day 0: (Day of transfection) Perform a visual assessment of confluence state of the cells. If cells are 65–75% confluent, you may proceed with the transfection procedure.

• 1. Replace medium on HEK293T cells—add 2 ml fresh FCS-containing DMEM (add medium VERY SLOWLY and CAREFULLY to the side walls of the wells, not directly to cells, or the cells will detach from the plate).

• 2. Make ScreenFect^®A transfection mix and incubate for 20 min at RT as per the manufacturer’s instructions. Transfection mix: dilute SFA reagent in SFA dilution buffer in tube A and dilute DNA in SFA dilution buffer in tube B. DNA dilution in Tube B: For each well of a 6 well plate, dilute 2.5 μg of retroviral plasmid DNA + 1.25 μg of pCL-Eco (encoding mouse-pseudotyped viral envelope) or pCl-10A1 (encoding human-pseudotyped viral envelope) in 240 μl of ScreenFect^®A dilution buffer.

• 3. After 20 min of RT incubation, add the SFA-DNA mix drop-wise and very carefully to HEK293T cells. Return cells to the incubator.

Day +1: Change medium—Replace medium (very carefully) on HEK293T cells.

Day +2: Collect 48 h virus supernatants.

• 1. Collect 48 h virus supernatants (sups) into 15 ml Falcon tubes and place sups at 4 °C Overnight.

• 2. Add 2 ml fresh serum-containing DMEM to each well and return HEK 293T cells to the incubator.

• 3. Optional: Plate target cells for infection after 18–24 h (Appendix 1.1.1.3).

Day +3: Collect 72 h virus supernatants.

• 1. Collect 72 h sups. Pool with 48 h virus supernatants.

• 2. Add polybrene to the (48 + 72) h supernatants to a final concentration of 8 μg/ml.

• 3. Filter through a 0.45 μm filter. Use supernatants immediately for infection of target cells (Appendix 1.1.1.3) or freeze aliquots at −20 °C. Note that the efficiency of virus transduction goes down exponentially with freeze-thawing.

1.3.2 Transduction of Target Cells with HMGB1-GFP-Expressing Retrovirus

Virus-mediated infection (transduction) by pCL-Eco or pCl-10A1-pseudotyped retrovirus has allowed us to generate HMGB1-GFP-expressing MEFs, DDCs, HFFs, and HeLa cells using the following protocol.

1.3.2.1 Material

• 1. DMEM supplemented with 10% heat-inactivated FCS, 1× NEAA, 1× penicillin–streptomycin, 1 mM sodium pyruvate, and 2 mM l-glutamine.

• 2. Cell culture incubator maintained at 37 °C with 10% CO₂.

• 3. Sterile flat bottom 12-well plates (Tissue culture-treated).

• 4. Pipette filter tips (20, 200, and 1000 μl).

• 5. Serological Pipettes (5 and 10 ml).

• 6. Mouse- or human-pseudotyped virus supernatants (filtered and polybrene-supplemented).

• 7. Target cells plated the day prior to retroviral transduction on 12-well plates.

• 8. Tabletop centrifuge equipped with a high-capacity microplate rotor and plate holders (preferably one that is capable of maintaining cells at 37 °C during spin infection).

• 9. Blasticidin-S hydrochloride (CAS 3513-03-9).

1.3.2.2 Methods

Day −1: Plate murine/human target cells∗ such that target cells are no more than 50–60% confluent at the time of transduction (there must be room on the plate for cells to divide, since it is during cell division that the retrovirus can access the cell nucleus and integrate into the host DNA).

∗MEFs and DDCs are plated at 20000 or 15,000 cells/well, respectively, HFFs are plated at 32000 cells/well of a 12-well plate.

Day 0: Virus-mediated transduction of target cells.

• 1. Apply 37 °C-warm supernatants (filtered + supplemented with polybrene to 8 μg/ml) to target cells (typically 2 ml of virus supernatant/well of a 12-well plate).

• 2. Perform a spin-infection at 2200 rpm (~900 × g), at 37 °C, for 90 min. We use an Eppendorf centrifuge model 5810 R equipped with the A-4-81-MTP/Flex high-capacity microplate rotor (16.3 cm radius) with four plate holders.

• 3. After 90 min spin-infection, return cells to the 37 °C incubator.

Day +2 (48 h after target cell transduction): detach cells from plate and passage into 6-well plates and, if desired to increase the frequency of transduced cells, initiate selection with blasticidin-S HCl-containing DMEM. For antibiotic selection of pM6pBLAST-transduced MEFs we use 10 μg/ml of blasticidin; murine DDCs, human Hs27 fibroblasts and HeLa cells require higher blasticidin concentrations (15–20 μg/ml).

As early as 48 h after retrovirus transduction, expression of the mHGMB1-GFP fusion protein in mouse or human cells can be assessed. The predominantly nuclear localization and the electrophoretic mobility pattern of the mHGMB1-GFP reporter in C57BL/6J MEFs are shown in Fig. 9 (Table 3).

Fig. 9

Expression of the mHMGB1-GFP retroviral reporter 48 h after transduction of C57BL/6J MEFs. (a) Merged phase contrast and green fluorescence images of transduced MEFs. (b) Western blot analysis of transduced (+) and mock-transduced (−) MEFs. Replica membranes were incubated with anti-human HMGB1 or anti-GFP antibodies. The higher molecular weight portions of the two membranes (100–150 kDa) were incubated with anti-human vinculin antibody as a loading control. The molecular mass of nuclear HMGB1 is ~25 kDa, whereas cytoplasmic and extracellular HMGB1 runs at ~30 kDa, largely due to acetylation. The multiple bands in the size range for the fusion protein likely reflect posttranslational modifications (acetylation, phosphorylation, and methylation) [25,26,27]

Appendix 2

See Fig. 10.

Fig. 10

A fixation-permissive method for detecting necrosis by flow cytometry. (a) Schematic representation of fixable LIVE/DEAD^® necrosis dyes. Live cells are dimly fluorescent, whereas dead cells, with a permeable membrane (indicated by the dashed line), display higher fluorescence. (b) Univariate histograms depicting differential staining by LIVE/DEAD^® dye of C57BL/6J DDCs left untreated or exposed to a cell death inducer. In the absence of LIVE/DEAD^® stain (“unstained” panel), untreated cells show no fluorescence. LIVE/DEAD^® stained, untreated cells are mostly found in the LIVE cell gate (left peak) while cells exposed to high levels (100 mM) of hydrogen peroxide (H₂O₂) which damages the permeability barrier, are found in the DEAD cell gate (right peak) of the histogram. Cells treated with 10 μM staurosporine (Sta) to induce apoptosis remain mostly within the LIVE cell gate because the permeability barrier is not broken. About 20% of cells stain heavily with the dye, presumably due to secondary necrosis after Sta-induced apoptosis. Treatment with both agents was for 3 h before preparation for FACS. Samples were analyzed by flow cytometry using 633 nm excitation and ~661 nm emission (FACScalibur cytometer, FL4 channel). (c) Proportion of necrotic (LIVE/DEAD^high) cells in samples shown in b. (d) The morphology and PI staining profile of C57B/L6J DDCs shown in b are consistent with their LIVE/DEAD staining prolife. Shown are merged images obtained from the combination of the PI (red) channel and the phase contrast channel microscopy analysis of control, 100 mM H₂O₂- or 10 μM Sta-treated cells. Changes in cell morphology range from a flat shape (untreated controls) to a round shape with enlarged nuclei and expanded cytoplasm (necrotic cells, with PI+ nuclei) to a wrinkling, stellate shape (apoptotic cells, a few PI+ nuclei)

Appendix 3: Macro

/∗ IJ macro to create ROIs for parasite, PV etc... by GabyGMartins @Instituto Gulbenkian Ciencia, Advanced Imaging Facility - v0.2 2019-01-02 macro assumes images are loaded with bioformats in imageJ/FIJI as a composite hyperstack and reading of metadata expects pixel sizes to be properly scaled, as measurements are performed in microns and that chromatic shift has been corrected - if there is pixel-shift then measurements might not be accurate ∗/ roiManager("reset"); run("Select None"); run("Make Composite"); run("Channels Tool..."); //activates channel tool and gives user a chance to turn off unwanted channels (eg bright field) waitForUser("turn off unwanted channels then OK"); //asks user to switch to PV channel and store in variable PVchannel PVchannel = getNumber("Which channel do you use to detect the PV?", 3); Stack.setChannel(PVchannel); /∗ Proceed to identifying semi-automatically the PV: activates wand tool so user can click on parasite adjusting the "tolerance" of the wand the ROI matches the shape of the PV a default tolerance value of 4500 work with our images; a different default can be inserted below to accelerate to process if magic wand fails user can also change ROI selection tool manually and draw the parasite ∗/ setTool("zoom"); //activates zoom function so user can zoom in on parasite waitForUser("Click on parasite to zoom in then click OK"); setTool("wand"); run("Wand Tool...", "tolerance=4500 mode=4-connected"); // user can change the default tolerance here waitForUser("Click inside the parasite to select and double click on wand tool to adjust tolerance"); run("Enlarge...", "enlarge=0.1"); //user here can change the area of the ring around PV // stores the ROIcontaining the PV to ROImanager roiManager("Add"); roiManager("Select", 0); roiManager("rename", "PV") //create band of cytoplasm outside of parasite and stores it as cytosol in ROI manager run("Make Band...", "band=1"); //change thickness of band here roiManager("Add"); roiManager("Select", 1); roiManager("rename", "cytosol") // create band of 0.8 μm representing the parasite's cortex and call it "PVM" roiManager("Select", 0); run("Enlarge...", "enlarge=-0.8"); run("Make Band...", "band=0.8"); roiManager("Add"); roiManager("Select", 2); roiManager("rename", "PVM") // prepare ROImanager for measurements roiManager("Select", newArray(0,1,2)); roiManager("Show All"); //activates several measurements and then stores measurements into a results table run("Set Measurements...", "area mean standard modal min shape feret's integrated stack display redirect=None decimal=3");roiManager("multi-measure measure_all");