Neurotoxic activities of culture fluids from HIV-1/VSV infected microglia attenuated by microglia-astrocyte co-cultivation.

<p>Mouse neurons were cultured for 10 d and exposed to the 20% conditioned media (CM) from uninfected or HIV-1/VSV infected microglia and/or astrocytes for 24 h. MAP-2 (green) and NeuN (red) expression by neurons was visualized by immunohistochemical staining. Characteristic neurotoxicity, including released nuclear materials and broken neurites, were observed in neurons treated with CM from HIV-1/VSV infected microglia alone. Percentages of apoptotic neurons were evaluated by the ratio of TUNEL⁺ (green) neurons to DAPI⁺ (blue) cells. The results are depicted as a mean percentage of apoptotic cells±SEM of three experiments. Significant reduction of percentage of apoptotic cells were observed in HIV-1/VSV infected co-cultures and astrocyte groups, compared with those of neurons cultured in microglial CM alone (n = 3 determines/group, p<0.01, determined by one-way ANOVA analysis and Tukey's multiple comparison post-hoc tests). Bars for MAP-2/NeuN, 20 µm; bars for DAPI and TUNEL, 50 µm.</p

Astrocyte effect on the proteome of HIV-1/VSV infected microglia.

a<p>Compared with uninfected microglia.</p>b<p>Theoretical isoelectric point calculated by Swissprot database at <a href="http://ca.expasy.org/sprot/" target="_blank">http://ca.expasy.org/sprot/</a>.</p>c<p>Number of peptides detected by mass spectrometry for each identified protein.</p

Immunohistochemical validation of proteomic profiling.

<p>Uninfected or HIV-1/VSV infected microglia were cultured for 24 h in the absence (MCG) or presence of astrocytes (MCG+AST). This arbitrary time point was chosen as it reflected dynamic changes in cell-cell interactions. Uninfected microglia cultured in the presence of 20 µg/ml TNF-α for 6 hours served as an activated microglia control. (A) Microglia were stained for the expression of the cytoskeletal proteins tubulin-α (green) and F-actin (red); the specific microglial marker ionized calcium binding adaptor molecule 1 (Iba-1) (red); and the activation indicator, vimentin (green). DAPI staining of nuclei shows total number of microglia. Arrowheads point to migratory morphologies. Scale bar, 20 µm; original magnification, ×63. Quantification of (A) tubulin-α, (B) F-actin, and (C) vimentin expression was performed via laser confocal microscopy and the ratio of overall fluorescence to cell numbers was calculated by digital image analysis using Image-Pro Plus version 5.1 software (Media Cybernetics, Inc.). Significant differences in mean fluorescence±SEM for n = 5 determinations/group was performed by one-way ANOVA and Tukey's post-hoc multiple comparisons where p<0.05 was considered significant.</p

BMM mobility after exposure to micoglia/astrocvte conditioned media.

<p>Migration of CMFDA-labeled BMM was assessed for 3 h using confocal microscopy live imaging in μ-slide chemotaxis assays comparing fresh media with conditioned media (CM) from microglia alone (MCG), astrocytes alone (AST) or microglia-astrocyte co-cultures (MCG+AST). Inhibitors wiskostatin (WISK, 20 µM) and latrunculin A (LatA, 0.2 µg/ml) were utilized to inhibit BMM migration. (A) Representative migrating BMM cultured in fresh medium (Medium) or CM fro microglia-astrocyte co-cultures (MCG+AST) were tracked (X's in different color tracings) and evaluated at 60 min, 120 min, and 180 min. Scale bar, 10 µm; original magnification, ×10. (B) Plots of each individual cell migration were generated from tracking data acquired for 3 h cultures in medium (Fresh Medium), CM from microglia (MCG), astrocytes (AST), or astrocyte-microglia co-cultures (CM), and in the presence of WISK and LatA inhibitors. Data were acquired by laser confocal microscopy and ImageJ software (NIH) interfaced with the ManulTrack plugin. (C) Migration velocities for each individual cell track were determined with the Chemotaxis and Tool software tool (ibidi). Significant differences in the mean±SEM migration velocities for n = 50 determinations/group were assessed by two-tail Student's t test where *p<0.05 was considered significant.</p

Ingenuity Pathway Analysis.

<p>Dynamic pathway/network modeling of the microglial proteome was afforded by integration of protein/gene data to the Ingenuity Pathway Analysis software of Ingenuity® Systems. Generated pathways show significant correlations with cellular assembly and organization (A) and cell death (B) networks. Interactions between proteins are shown by lines, whereas the lines with arrows represent direct interactions and no-arrow lines indicate binding only. Solid lines show the direct interaction, while the broken lines show indirect interaction. Nodes are represented by shapes and colors. Functions are indicated by shapes: diamonds for enzymes, squares for cytokines, rectangles for ligand dependant nuclear factors, triangles for kinases, ovals for transcription regulators, trapezoids for transporters, and circles indicate other interactions. Red color nodes represent the microglial proteins which are up-regulated upon HIV-1/VSV infection, while the green color nodes represent down-regulated proteins. Nodes without color represent proteins not input by user, but interpreted by the database as highly probable interactions within the network. The abbreviation used are: ACO1, aconitase; ACTB, beta-actin; ANXA1, annexin A1; ANXA2, annexin A2; APC, adenomatous polyposis coli protein; ATP2A2, sarcoplasmic reticulum 2+-Ca-ATPase; ATP6V1A, vacuolar ATP synthase catalytic subunit A; BLVRA, biliverdin reductase A; CAPRIN1, GPI-anchored protein p 137; COTL1, coactosin-like protein; DDX1, dead (Asp-Glu-Ala-Asp) box polypeptide 1; FABP4, fatty acid binding protein 4; FRMD4B, GRP1 binding protein; FTL1, ferritin light chain 1; GMFB, glia maturation factor beta; GSN, gelsolin; HSPA9, heat shock protein 70; LGALS3, galectin-3; LYZ, lysozyme; LMNA, lamin A; LMNB2, lamin B2; MARCKS (includes EG∶4082), myristylated alanine-rich protein kinase C substrate; MAPRE1, microtubule-associated protein; MSN, moesin; PDCD6IP, programmed cell death 6-interacting protein; MVP, major vault protein; NDUFS1, NADH dehydrogenase (ubiquinone) Fe-S protein 1; NNT, nicotinamide nucleotide transhydrogenase; PACSIN2, protein kinase C and casein kinase substrate in neurons protein 2; PLCB4, phospholipase C beta 4; PRDX1, peroxiredoxin-1; RDX, radixin; RPS14, 40S ribosomal protein S14; TPI1, triosephosphate isomerase; TPM3, tropomyosin-3; TUBA1C, alpha-tubulin 6; USP5, ubiquitin carboxyl-terminal hydrolase 5; VCP, transitional endoplasmic reticulum ATPase; VIL2, cytovillin; WASF2, Wiskott-Aldrich syndrome protein family member 2; YWHAZ, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta.</p

Infected mice lacking T cells develop cognitive impairment and have elevated number of monocytes/macrophages in the brain.

<p><b>A.</b> Control and EcoHIV-infected nude mice with or without ART were tested 24 h after fear conditioning training for contextual fear response and 24 h later for cued fear response. *P<0.05, **P<0.01, EcoHIV vs. EcoHIV+ART; #P<0.05, EcoHIV vs. PBS. <b>B.-D.</b> Flow cytometry analysis of macrophage or monocytes in brains of nude mice with EcoHIV, EcoHIV with cART or PBS showing representative dot plots. Cell populations were gated based on isotype control antibodies. <b>B.</b> Staining for CD45 and CD11b and negative for Ly-6G/C. <b>C.</b> Staining for CD45, CD11b and Ly-6C and negative for Ly-6G. <b>D.</b> As determined by flow cytometry using 5 mice per group, the number of cells in each population per total mouse brain are shown. <b>E.</b> Total and integrated vDNA was measured by QPCR and nested QPCR, respectively, in infiltrating leukocytes isolated from the brain of EcoHIV-infected mice with and without cART. *P<0.05, **P<0.01, ***P<0.001.</p

Both EcoHIV and MLV infect mouse brain but only EcoHIV-infected mice develop cognitive impairment.

<p><b>A.</b> Design of the experiment. <b>B.</b> Groups of mice were gavaged with cART or vehicle before and during infection with indicated viruses or vehicle. At day 21 after infection, mice were tested for errors (left panel) and latency (middle panel) in finding the hidden platform, the right panel shows latency to reach the visible platform. RT represents the retention trial. <b>C.</b> Two days after completion of RAWM, the same mice were used to test contextual response to fear conditioning training. The percentage of freezing time was calculated for contextual fear memory deficit in each group of mice. <b>D.</b> Mice were euthanized on day 40 after infection and levels of total vDNA in spleen and brain measured by QPCR (left panel) and integrated viral DNA measured by nested QPCR in PM (right panel). All data are mean ± standard errors. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 EcoHIV vs. EcoHIV+cART; ^#<i>P</i><0.05, ^##P<0.01, ^###<i>P</i><0.001 EcoHIV vs. MLV.</p

Features of EcoHIV-infected mice compared to HIV-infected people.

<p>Features of EcoHIV-infected mice compared to HIV-infected people.</p

EcoHIV infects mice and induces antiviral immune responses but not immunodeficiency in mice.

<p><b>A.-B.</b> Kinetics of production of total EcoHIV DNA (left panels), integrated EcoHIV DNA (middle panels), and genomic EcoHIV RNA (right panels) were measured by QPCR in <b>A.</b> spleen and <b>B.</b> PC at the times indicated after infection. Each point represents a single mouse. <b>C.</b> EcoHIV <i>gag</i> RNA burden at each time point in copies per ml of whole blood was measured at the times after infection indicated. The dashed line indicates limit of detection of 10 copies. <b>D.</b> The frequency of interferon-γ expression by CD4⁺ or CD8⁺ spleen cells at the time indicated after EcoHIV or mock infection was measured by flow cytometry. <b>E.</b> The number of CD4⁺ and CD8⁺ T cells was measured by flow cytometry at the indicated times and the CD4⁺:CD8⁺ ratio is shown. Symbols represent individual mice, the horizontal lines represent the mean. <b>F.</b> Longitudinal anti-NDK Gag antibodies in 3 EcoHIV-infected mice were measured by ELISA at the times indicated after infection. Each panel represents titers in a single mouse, mean +/- standard errors are shown.</p

EcoHIV establishes latent reservoirs in resting CD4⁺ cells that are inducible by epigenetic modulators.

<p><b>A.</b> CD4⁺ T cells isolated from spleen at day 25 post-infection were cocultured with uninfected cells in the presence or absence of ABC as indicated prior to vDNA amplification, each symbol represents culture from a single mouse. <b>B.</b> Six weeks after EcoHIV infection, CD4⁺ splenic T cells were harvested and purified from donor male 129X1/SvJ mice and injected into the recipient female 129X1 nude mice; their PM were collected after one week. Y chromosome and EcoHIV <i>gag</i> RNA levels in PM were determined by QPCR. Each symbol represents a single mouse. <b>C.-D.</b> Twelve weeks after infection, resting CD4⁺ T cells were purified from the spleen and <b>C.</b> The levels of integrated EcoHIV DNA and <b>D.</b> Genomic RNA were measured by QPCR. <b>E.</b> Eight weeks after EcoHIV infection of mice, resting CD4⁺ T cells were isolated and exposed to the agents indicated in culture for 2 days prior to collection and measurement of EcoHIV mRNA QPCR. <b>F.</b> Six weeks after infection by EcoHIV-Luc, mice were treated as shown, euthanized and splenic CD4⁺ cells isolated for measurement of virus RNA expression (left panel) or protein expression (right panel). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p