Cathepsin B is released from lysosomes in HIV-infected MDM.

<p>To analyze the lysosomal localization of cathepsin B, cathepsin B and LAMP2 immunoreactivity were assessed by <i>in situ</i> PLA (Duolink) in uninfected and HIV-infected MDM 3, 6 and 12 dpi. Cathepsin B colocalizes with LAMP2 in uninfected MDM (A, B and C; top panels). However, little colocalization is seen in HIV infected MDM (D, E and F; bottom panels). The presence of individual proteins was determined by immunofluorescence staining (G, H, I, J, K and L). As seen in the right panels both, cathepsin B (red) and LAMP2 (green) are expressed in uninfected (G, H, and I) and HIV-infected (J, K, L) cells. The results presented in this figure are representative of 3 experiments.</p

Post-mortem brain tissues received from the National NeuroAIDS Tissue Consortium.

<p>1)HIVE: HIV encephalitis.</p><p>2)HAD: HIV associated dementia.</p><p>3)MCMD: Minor cognitive motor disorder.</p

Expression of cathepsin B and cystatin B in the basal ganglia of post-mortem brain tissues.

<p>Basal ganglia tissue from HIV-seronegative (A, F and K) and HIV seropositive (B-E, G-J and L-O) patients were stained with mouse anti-human cathepsin B followed by Alexa 488 conjugate goat anti-mouse (green), or mouse anti-human cystatin B followed by Alexa 488 conjugate goat anti-mouse (green), and rabbit anti-human Iba-1 followed by Alexa 546 goat anti-rabbit (red), and nuclear staining by DAPI (blue). Unstained tissues were used as controls as illustrated in K to O. Magnification: 63×.</p

Effect of HIV infection on cathepsin B secretion in macrophages.

<p>Cell supernatants (n = 4) from HIV_ADA-infected (solid bars) and uninfected (open bars) macrophage cultures were collected, centrifuged, and tested for cathepsin B, cystatin B and cystatin C expression by antigen capture ELISA. (A) MDM secreted high levels of cathepsin B at all time points assayed. There was an increase in cathepsin B expression in the HIV-infected samples as compared with uninfected controls at 12 dpi (*p<0.05; A). HIV-infected and uninfected macrophages showed no differences in secretion of cystatin C or B (B and C). The ratio of cathepsin B to cystatin B and cathepsin B to cystatin C were calculated over time in culture (D). Cystatin B was present at higher concentrations than cathepsin B at all time points assayed, as indicated by the ratio of cathepsin B to cystatin B lower than 1. However, an increased cathepsin B to cystatin C ratio was observed in both HIV-infected and uninfected macrophages at all time points. At 12 dpi the ratio of cathepsin B to cystatin C in HIV-infected cells was higher in HIV-infected than uninfected cells (*p<0.05, D).</p

Increased cathepsin B mRNA after HIV infection.

<p>MDM from 8 different donors were inoculated with HIV-1_ADA or with serum-free media (uninfected controls) for 12 days and cell pellets collected at 3, 7, and 12 days post-infection. Changes in mRNA levels are shown as <i>Fold change</i>  = 2 ^ΔΔCt  = 2 ^{(Δ Ct control – Δ Ct experimental)} for cathepsin B (white), cystatin B (grey), and cystatin C (black). The mRNA levels of cystatins B and C remained similar after HIV infection. A significant increase in mRNA expression was found for cathepsin B in HIV infected MDM at 12 days compared to 3 (*p = 0.038, B) and 7 dpi (*p = 0.028).</p

Cathepsin B contributes to neuronal apoptosis caused by HIV-infected MDM.

<p>Apoptosis was measured using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay at 6 and 12 days post-infection as shown by green fluorescence in neurons stained with DAPI (blue) (Panel A). These results in panel A are representative of four experiments. The SK-N-SH cells exposed to MCM from uninfected MDM (1–2) with the cathepsin B antibody (3–4) do not show apoptosis. Neuronal apoptosis increased after exposure to HIV infected MCM (5–6) from later times post- infection. Inhibition of cathepsin B with the specific inhibitor CA-074 decreased the neuronal apoptosis in each of the time points (7–8). Pretreatment of medium with a monoclonal antibody against cathepsin B at 1∶500 dilution also decreased neuronal apoptosis (9–10). Quantitative analysis of staining ratio of apoptotic (green )/non-apoptotic (blue) nuclei using Image-based Tool for Counting Nuclei (ITNC) from Image J software (NIH) revealed a significant increment in percentage of apoptotic neurons at 12 dpi (p<0.05) compared to neurons treated with uninfected macrophage conditioned media. However, inhibition of cathepsin B by CA-074 decreased significantly the percentage of apoptotic neurons at 12 dpi (p<0.01) compared with neurons treated with HIV-infected media. MCM supernatant pre-treated with cathepsin B antibody reverted the percentage of apoptotic neurons at 12 dpi (p<0.01). Results in panel B represent the mean +/− SD of four biological replicates.</p

Secreted cathepsin B is more active in HIV-infected macrophages than in uninfected controls.

<p>Protein activity was measured by adding a synthetic peptide specific for cathepsin B conjugated to a red fluorogenic compound (RR₂-AFC) and read in a fluorometer at 400 nm excitation and 505 nm emission filters. Culture fluids from HIV-infected macrophages showed a significant increase in the activity of secreted cathepsin B at 3 and 12 days post infection (*p≤0.05). Results for cathepsin B activity are expressed as percentages of control (media only). Increased cathepsin B activity over the days after HIV infection (mean estimate increase/day 9.25 (SE 2.61), p = 0.002). The specificity of the assay is shown by the abrogation of any active cathepsin B after the addition of an inhibitor.</p

Cathepsin B does not interacts with cystatin C in HIV-infected MDM.

<p><i>In situ</i> PLA (Duolink) assay shows interaction between cathepsin B and cystatin C in uninfected cells (A, B and C; top panels) and decreased interactions in HIV infected MDM (D, E and F; bottom panels). Expression of cathepsin B (red) and cystatin C (green) was confirmed by immunofluorescence in uninfected (G and H; right top panels) and HIV-infected MDM (I and J; right bottom panels). This is a representative figure from 3 experiments performed.</p