HIV-1 reduces Abeta-degrading enzymatic activities in primary human mononuclear phagocytes

Abstract

The advent and wide introduction of antiretroviral therapy has greatly improved the survival and longevity of HIV-infected patients. Unfortunately, despite antiretroviral therapy treatment, these patients are still afflicted with many complications including cognitive dysfunction. There is a growing body of reports indicating accelerated deposition of amyloid plaques, which are composed of amyloid-β peptide (Aβ), in HIV-infected brains, though how HIV viral infection precipitates Aβ accumulation is poorly understood. It is suggested that viral infection leads to increased production and impaired degradation of Aβ. Mononuclear phagocytes (macrophages and microglia) that are productively infected by HIV in brains play a pivotal role in Aβ degradation through the expression and execution of two endopeptidases, neprilysin (NEP) and insulin-degrading enzyme. In this study, we report that NEP has the dominant endopeptidase activity toward Aβ in macrophages. Further, we demonstrate that monomeric Aβ degradation by primary cultured macrophages and microglia was significantly impaired by HIV infection. This was accompanied with great reduction of NEP endopeptidase activity, which might be due to the diminished transport of NEP to the cell surface and intracellular accumulation at the endoplasmic reticulum and lysosomes. Therefore, these data suggest that malfunction of NEP in infected macrophages may contribute to acceleration of β amyloidosis in HIV-inflicted brains, and modulation of macrophages may be a potential preventative target of Aβ-related cognitive disorders in HIV-affected patients.

Figure 1. Impaired clearance of phagocytosed monomeric Aβ in HIV-1 infected MDM

Primary cultured MDM were plated onto poly-D-lysine-coated 96-well plates (black walled for fluorescence measurement). MDM remained as uninfected (A-D) or infected with HIV-1_pYU-2 for 24 hours (E-H). 3 days later, cells were pulsed with monomeric Aβ42 (10μM) for 1 hour. Unbound Aβ was washed away and cells were fixed 4% paraformaldehyde after 24 hours. Immunofluorescence was conducted with Aβ-specific NU-2 mAb (green, C-D, G-H) and Hoechst 33342 for nuclear staining (blue, B, D, F, H). A and E, phase contrast images. D and H, merged images of B-C and F-G, respectively. Scale bar, 100 μm. I, Quantification of Aβ intensity was shown as Aβ/Hoechst 33342 fluorescence intensity % ratio (n=4 per group). ** denotes p<0.01 vs. Control group as determined by Student's t-test.

Figure 2. Comparison of IDE and NEP activities in MDM

Human monocytes were differentiated to MDM with M-CSF for 3 or 7 days, and cell lysates were collected for enzymatic assay with a fluorogenic substrate. Insulin (10 μM) or thiorphan (10 μM) was added into the reaction system to detect the IDE or NEP activity and the results were shown as shown as relative fluorescence units at 3 and 7 days of differentiation in vitro (DIV). ** denotes p<0.01 vs. control group as determined by t-test.

Figure 3. HIV-1 infection reduced NEP activity in MDM

Human MDM (2×10⁶ cells/well in 6-well plates) were infected (HIV group) or uninfected (Control group) with HIV-1_pYu2, incubated for 3, 7, or 10 days, and cell lysates were collected for immunoprecipitation and NEP activity determination shown as relative fluorescence units at 3, 7, and 10 days post infection (DPI). * denotes p < 0.05, and ** denotes p<0.01 by t-test assay.

Figure 4. HIV-1 infection of MDM

Human MDM were infected with HIV-1_pYu2 and fixed at 3 and 7 dpi, followed by immunofluorescence for HIV-1 p24 (green), phalloidin (red, staing F-actin), and Hoechst 33342 (blue, nuclear staining). (A-C), MDM at 3 dpi from different donors; (D), sham-infected MDM at 3 dpi; (E-G), MDM at 7 dpi different donors; (H), sham-infected MDM at 7 dpi.

Figure 5. HIV infection did not alter the expression of IDE/NEP in MDM

Cell lysates (25 μg/lane) from MDM with and without HIV-1 _pYu2 infection were subjected to SDS-PAGE and immunoblotting with anti-IDE (A), anti-NEP (C), or anti-β-actin control (A, C). B and D showed the quantitative comparisons of the IDE or NEP intensities between the control group and HIV group, respectively. NS denotes no statistical difference.

Figure 6. Confocal imaging of plasma membrane-localized and cellular NEPP in MDM

(A) Human MDM (2×10⁵ cells/cover slip in 24-well plates) were infected with HIV-1_pYu2 (HIV group) or with culture medium only (Control group) for 24 hours. Cells were fixed with 4% PFA at 7 dpi. Immunocytochemistry was carried without permeabilization to visualize surface-localized NEP (green) or permeabilization with 0.5% Triton-X-100 to stain intracellular NEP, followed by permeabilization and immunostaining of HIV-1 p24 (white), phalloidin (red), and Hoechst 33342 (blue). (B-D) Average immunofluorescent intensity per region of interest (ROI) was quantified for phalloidin (B), NEP (C), and NEP/phalloidin fluorescent intensity % ratio (n=15 per group). * and ** denotes p < 0.05 and 0.01 vs. Control group as determined by ANOVA and Turkey post hoc.

Figure 7. Laser-scanning Confocal Miscroscopy imaging of subcelluar NEP in MDM

Control and HIV-infected MDM were prepared on coverslips, fixed with 4% PFA, and permeabilized with 0.5% Triton-X-100. Then cells were immunostained with anti-NEP (green) plus anti-GRP94, anti-LAMP1, and anti-GM130 (red), which are organellar markers. Nuceli were visualized with Hoechst 33342 (blue). Images were taken with a confocal microscope at an objective of 40× and 2× zoom view for the regions of interest. Arrows indicate co-localized staining between red and green.

Figure 8. HIV-1 infection reduced Aβ degradation and NEP activity in microglia

(A) Human microglia (5×10⁴ cells/well in 96-well plates) were infected (HIV group) or uninfected (Control group) with HIV-1_pYu2 for three days, and tested for Aβ42 degradation at 1 and 5 uM concentrations as described in Fig. 1. The cells were subjected to immunofluorescence with anti-Aβ antibody (6E10) and Hoechst33342 for flurolometric quantification of Aβ oligomer fluorescent signal (Ex/Em=488 nm/519 nm) which was normalized by the nuclear staining signal (Ex/Em=350 nm/461 nm, n=5 per group). The results were presented as relative fluorescence ratio. * denotes p < 0.01 by t-test. (B) Human microglia (1×10⁶ cells/well in 6-well plates) were infected (HIV group) or uninfected (Control group) with HIV-1_pYu2 for three days, and cell lysates were collected for NEP activity determination shown as relative fluorescent units. * denotes p < 0.05 by Student's t-test assay.