Dysregulation of Macrophage-Secreted Cathepsin B Contributes to HIV-1-Linked Neuronal Apoptosis

Chronic HIV infection leads to the development of cognitive impairments, designated as HIV-associated neurocognitive disorders (HAND). The secretion of soluble neurotoxic factors by HIV-infected macrophages plays a central role in the neuronal dysfunction and cell death associated with HAND. One potentially neurotoxic protein secreted by HIV-1 infected macrophages is cathepsin B. To explore the potential role of cathepsin B in neuronal cell death after HIV infection, we cultured HIV-1ADA infected human monocyte-derived macrophages (MDM) and assayed them for expression and activity of cathepsin B and its inhibitors, cystatins B and C. The neurotoxic activity of the secreted cathepsin B was determined by incubating cells from the neuronal cell line SK-N-SH with MDM conditioned media (MCM) from HIV-1 infected cultures. We found that HIV-1 infected MDM secreted significantly higher levels of cathepsin B than did uninfected cells. Moreover, the activity of secreted cathepsin B was significantly increased in HIV-infected MDM at the peak of viral production. Incubation of neuronal cells with supernatants from HIV-infected MDM resulted in a significant increase in the numbers of apoptotic neurons, and this increase was reversed by the addition of either the cathepsin B inhibitor CA-074 or a monoclonal antibody to cathepsin B. In situ proximity ligation assays indicated that the increased neurotoxic activity of the cathepsin B secreted by HIV-infected MDM resulted from decreased interactions between the enzyme and its inhibitors, cystatins B and C. Furthermore, preliminary in vivo studies of human post-mortem brain tissue suggested an upregulation of cathepsin B immunoreactivity in the hippocampus and basal ganglia in individuals with HAND. Our results demonstrate that HIV-1 infection upregulates cathepsin B in macrophages, increases cathepsin B activity, and reduces cystatin-cathepsin interactions, contributing to neuronal apoptosis. These findings provide new evidence for the role of cathepsin B in neuronal cell death induced by HIV-infected macrophages

Microwave & magnetic proteomics of macrophages from patients with HIV-associated cognitive impairment

<div><p>Objective</p><p>HIV-infected monocytes can infiltrate the blood brain barrier as differentiated macrophages to the central nervous system, becoming the primary source of viral and cellular neurotoxins. The final outcome is HIV-associated cognitive impairment (HACI), which remain prevalent today, possibly due to the longer life-span of the patients treated with combined anti-retroviral therapy. Our main goal was to characterize the proteome of monocyte-derived macrophages (MDM) from HACI patients, and its association with their cognitive status, to find novel targets for therapy.</p><p>Methods</p><p>MDM were isolated from the peripheral blood of 14 HIV-seropositive women characterized for neurocognitive function, including: four normal cognition (NC), five asymptomatic (A), and five with cognitive impaired (CI). Proteins from macrophage lysates were isobaric-labeled with the microwave and magnetic (M2) sample preparation method followed by liquid chromatography-tandem mass spectrometry-based protein identification and quantification. Differences in protein abundance across groups classified by HACI status were determined using analysis of variance.</p><p>Results</p><p>A total of 2,519 proteins were identified with 2 or more peptides and 28 proteins were quantified as differentially expressed. Statistical analysis revealed increased abundance of 17 proteins in patients with HACI (p<0.05), including several enzymes associated to the glucose metabolism. Western blot confirmed increased expression of 6-Phosphogluconate dehydrogenase and L-Plastin in A and CI patients over NC and HIV seronegatives.</p><p>Conclusions</p><p>This is the first quantitative proteomics study exploring the changes in protein abundance of macrophages isolated from patients with HACI. Further studies are warranted to determine if these proteins may be target candidates for therapy development against HACI.</p></div

List of proteins identified by TMT labeling and differentially expressed among the HACI groups.

<p>List of proteins identified by TMT labeling and differentially expressed among the HACI groups.</p

Patient samples used for TMT labeling.

<p>Patient samples used for TMT labeling.</p

Differentially expressed proteins by TMT and mass spectrometry.

<p>A-D are proteins involved in the glycolysis pathway. E and F are the proteins involved with cell protection. G is the protein involved with neurotoxicity. H, and P are the proteins involved in oxidative stress and protein synthesis. From I to O are the proteins involved with the cell structure and motility. *p<0.05; **p<0.01.</p

Validation of proteins identified by western blot.

<p>(A) Proteins identified by TMT labeling were tested by western blot from MDM lysates of the same patients whose samples were used for proteomics. (B) Densitometry analyses for the western blots were normalized against GAPDH. The statistic analysis between the three groups of patients was performed using One-way ANOVA with a significance of *p<0.05. For Plastin-L, there were significant differences between NC vs CI (p = 0.0316), and between ANI vs CI (p = 0.042).</p

Predicted network of interactions between the proteins identified in macrophages from HACI patients.

<p>(A) Blue proteins are from our dataset and the grey colored proteins are the ones that connect the proteins in our dataset according to IPA software. (B) The lower panel shows the pattern of increase/decrease followed by vimentin, EEF1 and cathepsin B among the groups of HACI patients.</p

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

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