Preferential <i>in vitro</i> targeting of primary astrocytes by engineered lentiviruses sindMu-ZsGreen/GLAST IgG.

<div><p><b>A</b>. <i>In </i><i>vitro</i> targeting of astrocytes by lentiviruses – mixed primary glia cells isolated from the brains of one day old mice and were cultured <i>in </i><i>vitro</i>. Cultures were transduced with the indicated engineered lentiviruses, and 48h post transduction cells were harvested and stained for GFAP to specifically detect astrocytes. Cells were then analyzed by FACS for ZsGreen expression and for specific GFAP staining.</p> <p><b>Panel (a)</b> - transduction of cells with lentiviruses that did not incorporate IgG (sindMu-ZsGreen).</p> <p><b>Panel (b)</b> - transduction of cells with lentiviruses that incorporated an IgG isotype (sindMu-ZsGreen/IgG isotype).</p> <p><b>Panel (c)</b> - transduction of cells with lentiviruses that incorporated soluble GLAST IgG (sindMu-ZsGreen/GLAST IgG) and demonstrated preferential targeting for astrocytes.</p> <p><b>Panel (d)</b> - transduction of cells with lentiviruses that were pseudotyped with VSV-G and exhibited broad non-specific transduction to cells in the culture (VSV-G-ZsGreen).</p> <p><b>B</b>. FACS analysis demonstrating the composition of the mixed microglia culture. Cells of mixed microglia cultures were transduced with sindMu-ZsGreen/IgG isotype and stained for CD11b antigen as specific marker of microglia. Cells were then washed and analyzed for ZsGreen expression and CD11b staining by FACS.</p> <p><b>C</b>. A summary of quantitation of the <i>in </i><i>vitro</i> targeting of mixed microglia cell cultures. Cells were transduced with the indicated lentiviruses. 48h post transduction, cells were harvested and stained for astrocyte staining (GFAP) or microglia (CD11b). Cells were then analyzed by FACS for ZsGreen expression and for cell specific antigen. Values are presented relatively to the transduction efficiencies by the sindMu-ZsGreen/isotype IgG lentivirus. Results are representative of the means of triplicate wells; error bars show the standard deviation of the means.</p> <p><b>D</b>. Increasing amounts of the sindMu-ZsGreen/GLAST IgG lentivirus were used to transduce 3x10⁵ cells/well primary glia cultures. 48h post transduction, cells were harvested and stained for astrocyte staining (GFAP) or microglia (CD11b). Cells were then analyzed by FACS for ZsGreen expression and for cell specific antigen. Results are representative of the means of triplicate wells; error bars show the standard deviation of the means.</p></div

<i>In vivo</i> selective targeting of astrocytes by SindMu-ZsGreen/GLAST lentiviruses.

<div><p><b>A</b>. Broad-field images of brain sections following transduction with sindMu-ZsGreen/GLAST IgG lentivirus. The figure shows defined areas in the brain and cell types that were transduced and expressed viral-mediated ZsGreen. <b>panel a</b> - ZsGreen lentiviral expression; <b>panel b</b> - GFAP staining for astrocytes; <b>panel c</b> - NeuN staining for neurons; <b>panel d</b> - merged image for ZsGreen expression in neurons (NeuN) and astrocytes (GFAP). Indicated scale bar, <u>200µm (zoom x10)</u>.</p> <p><b>B</b>. Higher magnification of sindMu-ZsGreen/GLAST IgG transduction - engineered lentiviruses were injected into the hippocampus and thalamus of mice, and 14 days post injection brain slices were generated and stained with the appropriate antibodies. <b>panel a</b> - ZsGreen lentiviral expression; <b>panel b</b> - GFAP staining for astrocytes; <b>panel c</b> - NeuN staining for neurons; <b>panel d</b> – merged image for ZsGreen, GFAP and NeuN expression. Arrows present cells were GFAP and ZsGreen are co expressed. Indicated scale bar, 50µm (<u>zoom x40</u>).</p> <p><b>C</b>. Single cell imaging of the above transductions with sindMu-ZsGreen/GLAST IgG lentiviruses. Analysis shows preferential targeting of astrocytes in the CNS of mice by SindMu-ZsGreen/GLAST-1 IgG. <b>panel a</b> - ZsGreen lentiviral expression; <b>panel b</b> - GFAP staining for astrocytes; <b>panel c</b> – merged image for ZsGreen and GFAP expression at the level on a single cell. Indicated scale bar - 20µm (zoom x100).</p></div

<i>In</i><i>vivo</i> transduction with control sindMu-ZsGreen lentiviruses that display isotype IgG on their surface.

<div><p>To demonstrate specificity of the lentiviral targeting approach, <i>in </i><i>vivo</i> brain sections were transduced with sindMu-ZsGreen lentiviruses that displayed isotype IgG on their surfaces (sindMu-ZsGreen/ IgG isotype). <b>image a</b> - ZsGreen lentiviral expression; <b>image b</b> - GFAP staining for astrocytes; <b>image c</b> – NeuN staining for neurons; <b>image d</b> – merged image for ZsGreen, GFAP and NeuN expression. Indicated scale bar - 50µm(zoom x40).</p> <p><b>B</b>. Quantitation of in-vivo lentiviral targeting - Analysis of the relative percentage of ZsGreen-positive cells that also stained positively for NeuN or GFAP, following transduction with either VSV-G-ZsGreen or sindMu-ZsGreen/GLAST IgG recombinant lentiviruses. As shown, similar relative targeting index values for both astrocytes and neurons were observed when the VSV-G-ZsGreen was used. However, using sindMu-ZsGreen/GLAST IgG lentivirus, the NeuN-ZsGreen co-staining was significantly decreased, while the relative percentage of GFAP/ZsGreen-positive cells was significantly higher.</p></div

MG115 inhibits cathepsin C activity.

<p>(A) The cathepsin C assay was performed in the presence of 1 ng recombinant cathepsin C, the cathepsin C substrate Gly-Arg-AMC, and increasing concentrations of MG115 or the cathepsin C inhibitor Gly-Phe-DMK (GF-DMK). Cathepsin C activity was measured by analyzing Gly-Arg-AMC cleavage at 460 nm. (B) C57BL/6-derived macrophages were exposed to 2.5 mM LLOMe in the presence of increasing concentrations of MG115 or GF-DMK, and cell death was measured by PI exclusion two hours after LLOMe exposure.</p

Effect of proteasome inhibitors on cell death mediated by LLOMe and anthrax lethal toxin (LT).

<p>BALB/c-derived macrophages were exposed to 2.5 mM LLOMe (A) or anthrax lethal toxin (500 ng/ml PA and 250 ng/ml LF) (B) in the presence of increasing concentrations of the aldehyde proteasome inhibitors, MG115 and MG132, and the non-aldehyde proteasome inhibitor bortezomib. Cell death was measured by PI exclusion two hours after LLOMe/LT exposure. The above data is a representative experiment performed in triplicate.</p

The proteasome inhibitor MG115 blocks LLOMe-mediated lysosome rupture.

<p>(A) Flow cytometry analysis of LLOMe-, alum- and LT-treated cells. BALB/c-derived macrophages were exposed to 2.5 mM LLOMe, LT (500 ng/ml PA and 250 ng/ml LF), or alum (150 µg/ml) in the absence and presence of 100 µM MG115 and 50 µM bortezomib. Lysosome and membrane integrity were measured using LysoTracker and PI by flow cytometry 2 hours post LLOMe and LT challenge, and 6 hours post alum challenge. The flow cytometry plots are representative images of two experiments each performed in triplicate. (B) C57BL/6-derived macrophages were exposed to 2.5 mM LLOMe in the absence and presence of 100 µM MG115. Analysis of lysosome integrity was determined by acridine orange (AO) staining 2 hours post LLOMe exposure. The above data is representative of three experiments.</p

A Proteolytic Cascade Controls Lysosome Rupture and Necrotic Cell Death Mediated by Lysosome-Destabilizing Adjuvants

<div><p>Recent studies have linked necrotic cell death and proteolysis of inflammatory proteins to the adaptive immune response mediated by the lysosome-destabilizing adjuvants, alum and Leu-Leu-OMe (LLOMe). However, the mechanism by which lysosome-destabilizing agents trigger necrosis and proteolysis of inflammatory proteins is poorly understood. The proteasome is a cellular complex that has been shown to regulate both necrotic cell death and proteolysis of inflammatory proteins. We found that the peptide aldehyde proteasome inhibitors, MG115 and MG132, block lysosome rupture, degradation of inflammatory proteins and necrotic cell death mediated by the lysosome-destabilizing peptide LLOMe. However, non-aldehyde proteasome inhibitors failed to prevent LLOMe-induced cell death suggesting that aldehyde proteasome inhibitors triggered a pleotropic effect. We have previously shown that cathepsin C controls lysosome rupture, necrotic cell death and the adaptive immune response mediated by LLOMe. Using recombinant cathepsin C, we found that aldehyde proteasome inhibitors directly block cathepsin C, which presumably prevents LLOMe toxicity. The cathepsin B inhibitor CA-074-Me also blocks lysosome rupture and necrotic cell death mediated by a wide range of necrosis inducers, including LLOMe. Using cathepsin-deficient cells and recombinant cathepsins, we demonstrate that the cathepsins B and C are not required for the CA-074-Me block of necrotic cell death. Taken together, our findings demonstrate that lysosome-destabilizing adjuvants trigger an early proteolytic cascade, involving cathepsin C and a CA-074-Me-dependent protease. Identification of these early events leading to lysosome rupture will be crucial in our understanding of processes controlling necrotic cell death and immune responses mediated by lysosome-destabilizing adjuvants.</p></div

The proteasome inhibitor MG115 blocks LLOMe-mediated lysosome rupture.

<p>(A) Flow cytometry analysis of LLOMe-, alum- and LT-treated cells. BALB/c-derived macrophages were exposed to 2.5 mM LLOMe, LT (500 ng/ml PA and 250 ng/ml LF), or alum (150 µg/ml) in the absence and presence of 100 µM MG115 and 50 µM bortezomib. Lysosome and membrane integrity were measured using LysoTracker and PI by flow cytometry 2 hours post LLOMe and LT challenge, and 6 hours post alum challenge. The flow cytometry plots are representative images of two experiments each performed in triplicate. (B) C57BL/6-derived macrophages were exposed to 2.5 mM LLOMe in the absence and presence of 100 µM MG115. Analysis of lysosome integrity was determined by acridine orange (AO) staining 2 hours post LLOMe exposure. The above data is representative of three experiments.</p

CA-074-Me and Cathepsin C deficiency blocks cell death and protein degradation mediated by LLOMe.

<p>(A) J774A.1 macrophages, and wild type, cathepsin B- or C-deficient C57BL/6-derived macrophages were treated with 2.5 mM LLOMe, and cell death was measured by PI exclusion two hours after LLOMe exposure. Corresponding lysates were subjected to immunoblotting and were probed with anti-caspase-1 and actin antibodies (lower panel). (B) The <i>in vitro</i> assays were performed in the presence of recombinant cathepsin B or cathepsin C, the corresponding cathepsin B and C substrates, and increasing concentrations (0.01, 0.1, 1 and 10 µM) of MG132, the cathepsin C inhibitor GF-DMK or the cathepsin B inhibitor CA-074-Me. Cathepsin B and C activity was measured by analyzing Gly-Arg-AMC and Arg-Arg-AMC cleavage at 460 nm, respectively.</p

Expression of scFvFc on the surface of lentivirus transduced cells.

<p><i>Panel a</i> 293T cells were transduced with increasing dilutions (different MOIs as indicated) of lentivirus encoding the PS11-scFvFc-CD28-gp41-IRES-ZsGreen. Transduced cells were harvested, stained for Fc-surface expression, and analyzed by FACS. Expression of ZsGreen was measured to monitor levels of transduction. The graphs depict the percentage of transduced cells that express ZsGreen (blue diamonds) and the percentage values of transduced cells that express PS11-scFvFc as monitored by staining with APC-conjugated anti-human Fc IgG (pink squares). <i>Panels b</i> and <i>c</i>, cell -surface expressed scFvFc proteins are functional. 293T cells were transduced with a lentivirus encoding PS11-scFv-Fc-CD28-gp41-IRES-ZsGreen (<i>Panel b</i>) or 11A-scFvFc-CD28-gp41-IRES-ZsGreen (<i>Panel c</i>). Cells were incubated with biotinylated GD03-Fc, a specific antigen for 11A-scFvFc, and stained for streptavidin-APC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003181#s4" target="_blank">Methods</a>, and then analyzed by FACS. Note that two clusters of cells in <i>Panel c</i> represent high (R2 gated) and low (R3 gated) levels of 11A-scFvFc on their surface as measured by APC staining. These could reflect variations in cell-surface expression levels resulting from multiple integration events of the scFvFc cassette following transduction, or the difference in transgene integration site, <i>i.e.</i>, its proximity to active transcriptional units. R2 = 1390 and R3 = 220 are MFI values of 11A scFvFc expressing cells, where percentage of positive cells in each gate is 31% and 49% respectively. <i>Panel d.</i> a summary of specific antigen binding by the scFvFc displayed on the lentivirus transduced cells. The table shows the percentage of transduced cells expressing X48-scFvFc-CD28-gp41 or PS11 scFvFc-CD28-gp41 that bind to their cognate or irrelevant biotinylated antigens as visualized by APC staining and their corresponding MFI values.</p