HIV-1 Reduces Aβ-Degrading Enzymatic Activities in Primary Human Mononuclear Phagocytes

This information is current as Xiqian Lan, Jiqing Xu, Tomomi Kiyota, Hui Peng, Jialin C. of September 26, 2021. Zheng and Tsuneya Ikezu J Immunol 2011; 186:6925-6932; Prepublished online 6 May 2011; doi: 10.4049/jimmunol.1100211

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

HIV-1 Reduces Ab-Degrading Enzymatic Activities in Primary Human Mononuclear Phagocytes

Xiqian Lan,* Jiqing Xu,* Tomomi Kiyota,* Hui Peng,* Jialin C. Zheng,* and Tsuneya Ikezu*,†,‡

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-b (Ab), in HIV-infected brains, though how HIV viral infection precipitates Ab accumulation is poorly understood. It is suggested that viral infection leads to increased production and impaired degradation of Ab. Mononuclear phagocytes ( and microglia) that are productively infected by HIV in brains play a pivotal role in Ab degradation through the expression and execution of two endopeptidases, (NEP) and insulin-degrading enzyme. In this study, we Downloaded from report that NEP has the dominant endopeptidase activity toward Ab in macrophages. Further, we demonstrate that monomeric Ab degradation by primary cultured macrophages and microglia was significantly impaired by HIV infection. This was accom- panied 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 . Therefore, these data suggest that malfunc- tion of NEP in infected macrophages may contribute to acceleration of b amyloidosis in HIV-inflicted brains, and modulation of macrophages may be a potential preventative target of Ab-related cognitive disorders in HIV-affected patients. The Journal of http://www.jimmunol.org/ Immunology, 2011, 186: 6925–6932.

ntiretroviral therapy has shown significant advancement (HAART), which is probably the result of a number of adverse in the longevity of HIV-infected patients. The affected effects inflicting HAART-medicated patients. These adverse ef- A populations are facing new challenges of age-related fects are known as AD risk factors, such as immune reconstitution complications, including diabetes, , atherosclerosis, psy- syndrome, lypodystrophic and metabolic effects causing hyper- chiatric disorders, and cognitive dysfunctions leading to senile lipidemia, alterations in body fat distribution to metabolically

dementia (1–3). The pathogenesis of HIV-related brain injury may inactive areas, diabetes, and coronary artery disease (5–7). How- by guest on September 26, 2021 intersect with Alzheimer’s disease (AD) in several aspects, in- ever, how HIV infection or HIV infection in conjunction with cluding accumulation of amyloid-b peptide (Ab) in the brain, HAART medication precipitates Ab accumulation is largely un- activation of brain mononuclear phagocytes, dysfunction of the known. blood–brain barrier, glial inflammation, and metabolic disorders A significant body of evidence suggests that Ab accumulates in (4). Meanwhile, there is a growing body of evidence showing that AD are the result of an imbalance between Ab production and Ab brain amyloid deposition is increased in HIV-infected patients, clearance (8–10). Normally, Ab, generated via sequential pro- though the extent and relation of this deposition to the clinical teolysis of amyloid precursor (APP), in the CNS can be state and regional HIV infection remain unresolved (1). In addi- cleared via -mediated proteolysis or efflux to general tion, it has been reported that Ab amyloidosis is accelerated in the circulation through the neurovascular systems (for review, see brains of patients undergoing highly active antiretroviral therapy Refs. 8, 11, 12). Macrophages and microglia, which are the pre- dominant cells productively infected with HIV in the brain (13, 14), have been shown to play major roles in internalization and *Department of Pharmacology and Experimental Neuroscience, University of degradation of Ab (15, 16). Macrophages, the peripheral coun- Nebraska Medical Center, Omaha, NE 68198; †Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA terpart to microglia, have attracted much attention recently based 02118; and ‡Department of Neurology, Boston University School of Medicine, Bos- on the findings that a subset of microglia surrounding and at- ton, MA 02118 tempting to clear Ab-containing plaques in transgenic AD mouse Received for publication January 26, 2011. Accepted for publication April 12, 2011. models are bone marrow derived (17–19). Bone marrow-derived This work was supported in part by National Institutes of Health Grants microglia, but not resident microglia, have been reported to pre- R01MH083523, R21 AG032600, R01MH072539 (to T.I.), and P01 NS043985 (to T.I. and J.C.Z.). vent the formation of and even eliminate brain amyloid deposits Address correspondence and reprint requests to Dr. Tsuneya Ikezu, Boston University (18). However, there is little known about how HIV infection School of Medicine, 72 East Concord Street, L-606B, Boston, MA 02118. E-mail affects the capability of macrophages to catabolize Ab. It is sug- address: [email protected] gested that HIV infection together with normal aging can accel- Abbreviations used in this article: Ab, amyloid-b peptide; AD, Alzheimer’s disease; erate amyloid accumulation by both increasing Ab production ApoE, apolipoprotein E; APP, amyloid precursor protein; dpi, day postinfection; ER, endoplasmic reticulum; Grp94, heat shock protein 90 kDa b; HAART, highly active and impairing Ab degradation (3). Further, proinflammatory cyto- antiretroviral therapy; IDE, insulin-degrading enzyme; LAMP1, lysosomal-asso- kines, which are upregulated in the HIV-infected brains, can ciated membrane protein 1; MDM, monocyte-derived ; NEP, neprilysin; stimulate neurons to promote Ab production (20–23). NIH, National Institutes of Health; PFA, paraformaldehyde; ROI, region of interest. In contrast, HIV infection may cause a faulty Ab degradation by Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 macrophages and microglia, contributing to Ab accumulation. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100211 6926 HIV-1 REGULATION OF NEPRILYSIN IN MACROPHAGES

FIGURE 1. Impaired clearance of phagocytosed monomeric Ab 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-1pYU-2 for 24 h (E–H). Three days later, cells were pulsed with monomeric Ab42 (10 mM) for 1 h. Unbound Ab was washed away, and cells were fixed with 4% PFA after 24 h. Immunofluorescence was conducted with Ab-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 and C and F and G, respectively. Scale bars, 100 mm. I, Quantification of Ab intensity was shown as Ab/Hoechst 33342 fluorescence intensity percent ratio (n = 4 per group). **p , 0.01 versus control group as determined by Student t test. Downloaded from

Macrophages and microglia express two major Ab-degrading Medium A containing DMEM (Invitrogen, Carlsbad, CA) supplemented endopeptidases, namely neprilysin (NEP; also called neutral en- with 10% heat-inactivated human serum, 2 mM L-glutamine (2 mM), dopeptidase and enkephalinase, CD10, EC 3.4.24.15) (24, 25) and gentamicin (50 mg/ml), ciprofloxacin (10 mg/ml), and M-CSF (1000 U/ml; R&D Systems, Minneapolis, MN). Monocytes were cultivated in Medium insulysin (insulin-degrading enzyme [IDE], EC 3.4.24.56) (26). A for 7 d, allowing their differentiation into macrophages, which were NEP appears to be the predominant protease that degrades Ab in then referred to as monocyte-derived macrophages (MDM). MDM were the brain (27–30). NEP is a type II membrane-bound zinc metal- maintained in Medium B, which is Medium A without M-CSF. Human http://www.jimmunol.org/ loendopeptidase localized primarily on the plasma membrane with microglia were isolated following the described protocols (39, 41, 42). Fetal brain tissue (gestational age 14–16 wk) was obtained from the Birth its catalytic site exposed extracellularly (hence an ectopeptidase), Defects Laboratory, University of Washington (Seattle, WA), in full making this peptidase a prime candidate for peptide degradation at compliance with the ethical guidelines of the National Institutes of Health extracellular sites of amyloid accumulation. Numerous studies (NIH) and the Universities of Washington and Nebraska Medical Center. have implicated NEP as a rate-limiting Ab-degrading enzyme in At least three different donors were tested for the experiments using MDM the brain (25, 31, 32). Expression levels of the Ab-binding scav- and microglia. enger receptors scavenger receptor A, CD36, and receptor for Viral infection

advanced- end products and IDE, neprilysin, and by guest on September 26, 2021 MDM cultured in plates or on coverslips (intended for cell staining) were matrix metalloprotease-9 are decreased 2–5-fold in microglia in rinsed twice with 13 PBS, then incubated for 24 h with either Medium B the aged brain of an AD mouse compared with their littermate alone in the case of the control group or with Medium B containing HIV- controls (33). NEP and IDE become inactivated and downregu- 1pYu2 (titrated as 1 pg HIV p24 protein/cell; NIH AIDS Research and lated during the early stages of both AD and aging (34–36). Reference Reagent Program, Germantown, MD) in the infected group (43). Then cells were washed three times with 13 PBS and replaced with It was reported that HIV viral protein Tat-derived peptide in- Medium B, monitored for cytopathic effects by nuclear staining of apo- hibited NEP activity in vitro, and recombinant Tat added directly ptotic cells, and maintained for 3, 7, or 10 d. to brain cultures resulted in a 125% increase in soluble Ab (37, 38). Ab degradation in MDM and microglia Nevertheless, HIVencodes at least nine , so it will be more pathophysiologically relevant to examine the effect of the com- Primary cultured MDM or microglia were plated onto 96-well plates. Viral plete HIV viral infection on macrophage- and microglia-mediated infection was carried out as above. Briefly, solid Ab peptide (Ab 1-42; Ab catabolism. In this report, we examined whether Ab clearance Invitrogen) was dissolved in cold hexafluoro-2-propanol (Sigma-Aldrich, via degradation and the enzymatic activity of NEP in mononuclear phagocytes, both brain perivascular macrophages and microglia, is altered as a result of HIV infection. Our in vitro study demon- strates that HIV infection significantly impaired Ab degradation in mononuclear phagocytes. Further, we confirmed that NEP exhibits the dominant endopeptidase activity toward Ab degradation in mononuclear phagocytes. However, we found that HIV viral in- fection dramatically compromised NEP activity, but not NEP expression level, which was concomitant with significant re- duction in the cell-surface level of NEP protein and intracellular NEP accumulation in the endoplasmic reticulum (ER) and lyso- somes.

FIGURE 2. Comparison of IDE and NEP activities in MDM. Human Materials and Methods monocytes were differentiated to MDM with M-CSF for 3 or 7 d, and cell Cell culture lysates were collected for enzymatic assay with a fluorogenic substrate. Isolation and cultivation of monocytes were performed as previously Insulin (10 mM) or thiorphan (10 mM) was added into the reaction system reported (39). Briefly, human monocytes were recovered from PBMCs of to detect the IDE or NEP activity, and the results were shown as shown HIV-1 and hepatitis B seronegative donors after leukopheresis and purified as relative fluorescence units at 3 and 7 d of differentiation in vitro (DIV). by countercurrent centrifugal elutriation (40). Monocytes were cultured in **p , 0.01 versus control group as determined by t test. The Journal of Immunology 6927

well plates (Thermo Scientific) overnight at 4˚C. The plates were washed six times with PBS containing 0.5% Tween-20, and nonspecific binding of Ab was blocked by incubation with 1% PBS-BSA (Sigma-Aldrich) for 3 h at room temperature. Plates were then washed six times with PBS con- taining 0.5% Tween-20. MDM lysates (200 mg) or microglia lysates (100 mg) were added to the coated plates and incubated at 4˚C overnight. After washing six times, the fluorogenic peptide (10 mM) diluted in reaction buffer (100 mM Tris-Cl [pH 7.5], 50 mM NaCl, and 10 mM ZnCl2) was added and incubated at 37˚C in the dark for 1 h, then fluorescence was measured with excitation at 320 nm and emission at 405 nm. Effect of HIV viral proteins on endopeptidase activities of FIGURE 3. HIV-1 infection reduced NEP activity in MDM. Human recombinant IDE and NEP MDM (2 3 106 cells/well in six-well plates) were infected (HIV group) or uninfected (Control group) with HIV-1pYu2, incubated for 3, 7, or 10 d, cell HIV-1 Tat protein (catalog number 2222) and HIV-1 gp120 (catalog number lysates were collected for immunoprecipitation, and NEP activity de- 7363) were obtained from the NIH AIDS Research and Reference Reagent termination shown as relative fluorescence units at 3, 7, and 10 dpi. *p , Program. Tat protein (0.1, 1, and 10 mg/ml) or gp120 (1, 10, and 100 mM) 0.05, **p , 0.01 by t test assay. was mixed with 20 ng recombinant human IDE (R&D Systems) or 10 ng recombinant human NEP (R&D Systems) in reaction buffer with shaking at room temperature for 10 min. This was followed by the addition of 10 St. Louis, MO) and incubated at room temperature for 1 h. The hexafluoro- mM fluorogenic peptide as described and incubated at 37˚C in the dark for 2-propanol was then removed by evaporation, and the resulting peptide 1 h. Fluorescence was measured with excitation at 320 nm and emission at 2 was stored as a film at 20˚C. The resulting film was dissolved in an- 405 nm. Downloaded from hydrous DMSO (Sigma-Aldrich) at 250 mM and then diluted at 10 mMin Medium B. MDM were infected with HIV-1pYu-2, and at 3 d postinfection Immunoblotting (dpi), the cells were incubated with 10 mMAb for 1 h, then fixed with Macrophage lysates were prepared as mentioned above. Protein concen- freshly depolymerized 4% paraformaldehyde (PFA) for 15 min. Standard trations were determined using the BCA Protein Assay Kit (Thermo Sci- immunofluorescence was performed using anti-Ab–specific Abs NU-2 entific). Protein (25 mg) was electrophoresed on 10% SDS-PAGE and (1:100 dilution; mouse mAb kindly provided by Dr. William Klein, transferred to Immuno-Blot PVDF membranes (Bio-Rad, Hercules, CA), Northwestern University, Evanston, IL) or 6E10 (1:1000 dilution, mouse; followed by incubation in blocking buffer (5% skim milk in TBST) for 1 h Covance Research Product, Princeton, NJ) and Alexa Fluor 488-conju- http://www.jimmunol.org/ at room temperature. Membranes were then incubated overnight at 4˚C gated anti-mouse IgG (H+L) (Molecular Probes/Invitrogen) secondary Ab. with primary Abs against IDE (1:500 dilution; rabbit polyclonal, PC730; Cells were counterstained with Hoechst 33342 (1:2000 dilution; Invi- EMD Chemicals, Gibbstown, NJ), NEP (1:100 dilution; mouse mono- trogen) and subjected for immunofluorescence microscopy and fluorescent clonal, NCL-CD10-270; Novocastra/Leica Microsystems, Bannockburn, intensity measurement. IL), and b-actin (1:40,000 dilution; mouse monoclonal, AC-15; Sigma- Cellular NEP and IDE endopeptidase activity assay Aldrich). Subsequently, membranes were incubated with HRP-conjugated secondary anti-rabbit or anti-mouse secondary Abs (1:10,000 dilution; MDM maintained in Medium B for 3–7 d were rinsed twice with ice-cold Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at room PBS and then lysed in lysis buffer (20 mM Tris-Cl [pH 7.4], 0.5% Trition temperature. Immunoreactive bands were visualized by enhanced chemi- X-100, 10% sucrose, 1 mg/ml aprotinin, and 10 mM phenylmethane sul- luminescence (GE Healthcare Biosciences, Piscataway, NJ) and captured fonyl fluoride; all from Sigma-Aldrich). The lysates were centrifuged at with CL-X Posure Film (Pierce). For data quantification, the films were by guest on September 26, 2021 15,000 3 g for 15 min at 4˚C, and protein concentrations of the super- scanned with a CanonScan 9950F scanner (Canon USA, Lake Success, natants were measured by standard BCA assay using the BCA Protein NY); the acquired images were then analyzed on a Macintosh computer Assay Kit (Thermo Scientific, Rockford, IL). using the public domain NIH image program (http://rsb.info.nih.gov/nih- Endopeptidase activities were assayed in a 96-well plate. In each well, 10 image/). mg lysate and 10 mM fluorogenic peptide Mca-RPPGFSAFK(Dnp)-OH (R&D Systems) were mixed in reaction buffer (100 mM Tris-Cl [pH Immunofluorescence 7.5], 50 mM NaCl, and 10 mM ZnCl ) and incubated in the dark for 1 h at 2 MDM were seeded onto 15-mm coverslips housed in 24-well plates. Seven 37˚C. Insulin (10 mM; Invitrogen), a potent competitive inhibitor of IDE dpi, cells were fixed with 4% PFA (Sigma-Aldrich), and immunocyto- toward Ab, or thiorphan (10 mM; Sigma-Aldrich), a specific NEP in- chemistry was conducted as reported (44). Briefly, to visualize plasma hibitor, was added to distinguish specific enzyme activities in the cell membrane-localized NEP, fixed cells were not permeabilized prior to in- lysates. Fluorescent intensity of the cleaved fragments was measured by cubation with blocking buffer containing 5% BSA and 5% normal donkey fluorometry with excitation at 320 nm and emission at 405 nm. IDE ac- serum in PBS for 1 h at room temperature. Afterwards, samples were tivity was defined as the activity sensitive to insulin inhibition, whereas incubated overnight with p24 Ab (1:50 dilution in PBS; mouse mono- NEP activity was defined as the activity sensitive to thiorphan inhibition. clonal, M0857; Dako North America, Carpinteria, CA) and NEP Ab (1: 50 Immunocapture-based NEP endopeptidase activity assay dilution in PBS; goat polyclonal; R&D Systems) at 4˚C, followed by washing with PBS three times. Then samples were incubated for 30 min at Goat anti-human NEP Ab (2 mg/ml) (AF1182; R&D Systems) diluted in room temperature with appropriate fluorescent-labeled secondary Abs (for sodium bicarbonate (100 mM [pH 9]) was coated on Nunc MaxiSorp 96- p24, Alexa Fluor 647 donkey anti-mouse IgG; for NEP, Alexa Fluor 488

FIGURE 4. HIV-1 infection of MDM. Human MDM were infected with HIV-1pYu2 and fixed at 3 and 7 dpi, followed by immunofluores- cence for HIV-1 p24 (green), phal- loidin (red, staining F-actin), and Hoechst 33342 (blue, nuclear stain- ing). MDM at 3 dpi from different donors (A–C); sham-infected MDM at 3 dpi (D); MDM at 7 dpi different donors (E–G); and sham-infected MDM at 7 dpi (H). Original mag- nification 3200. 6928 HIV-1 REGULATION OF NEPRILYSIN IN MACROPHAGES donkey anti-goat IgG) diluted 1:500 in PBS. After washing with PBS three times, cells were also incubated for 20 min at room temperature with Alexa Fluor 594-conjugated phalloidin (1:40; Invitrogen) to visualize F-actin cytoskeleton and with Hoechst 33342 (1:5000; Invitrogen) to stain nu- clear DNA. Images were acquired using a laser-scanning confocal mi- croscope (510 Meta; Carl Zeiss MicroImaging, Thornwood, NY) at an objective of 340 and a 32 zoom view for the interested area. To stain NEP within all cellular compartments, cell staining followed the above proce- dures except that cells were permeabilized with 0.5% Triton X-100 for 15 min at room temperature prior to blocking for nonspecific binding. The average fluorescence intensity for NEP, phalloidin, and Hoechst 33342 was measured per region of interest (ROI) for each group (15 fields per group), and the intensity value was normalized by the number of cells as de- termined by the Hoechst 33342 staining as per 100 cells. Normalized NEP and phalloidin intensity value of the same ROI was used for the calculation of NEP/phalloidin ratio for the group comparison. FIGURE 5. HIV infection did not alter the expression of IDE/NEP in For immunofluorescence of subcellular NEP, control and HIV-infected MDM. Cell lysates (25 mg/lane) from MDM with and without HIV-1 pYu2 MDM on coverslips were prepared as above and fixed with 4% PFA, infection were subjected to SDS-PAGE and immunoblotting with anti-IDE followed by incubation with 0.5% Triton X-100 for 15 min at room (A), anti-NEP (C), or anti–b-actin control (A, C). B and D show the temperature for membrane permeabilization. Then cells were incubated quantitative comparisons of the IDE or NEP intensities between the control with blocking buffer (0.1% Triton X-100, 5% BSA, and 5% normal donkey group and HIV group, respectively. serum in PBS) for 1 h at room temperature. To locate NEP at a subcellular level, double immunofluorescence was conducted by coincubating cells Downloaded from with NEP Ab (1:50; goat polyclonal; R&D Systems) and an Ab for lysosomal-associated membrane protein 1 (LAMP1), lysosomal marker established organelle markers, heat shock protein 90 kDa b (Grp94), ER (1:100; rabbit polyclonal, ab24170; Abcam), GM130, cis-Golgi matrix marker (1:100, rabbit polyclonal, ab3674; Abcam, Cambridge, MA), protein, and marker (1:100; rabbit monoclonal, ab52649; http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. Confocal imaging of plasma membrane-localized and cellular NEPP in MDM. A, Human MDM (2 3 105 cells/cover slip in 24-well plates) were infected with HIV-1pYu2 (HIV group) or with culture medium only (Control group) for 24 h. Cells were fixed with 4% PFA at 7 dpi. Immunocy- tochemistry was carried without permeabilization to visualize surface-localized NEP (green) or permeabilization with 0.5% Triton X-100 to stain in- tracellular NEP, followed by permeabilization and immunostaining of HIV-1 p24 (white), phalloidin (red), and Hoechst 33342 (blue). Scale bar, 50mm. Average immunofluorescent intensity per ROI was quantified for phalloidin (B), NEP (C), and NEP/phalloidin (D) fluorescent intensity percent ratio (n =15 per group). *p , 0.05, **p , 0.01 versus control group as determined by ANOVA and Tukey post hoc. The Journal of Immunology 6929

Abcam). Subsequently, cells were incubated with appropriate fluorescent- significant difference as determined by fluorescent intensity labeled secondary Abs diluted 1:500 in PBS (for NEP, Alexa Fluor 488 measurement (Fig. 1I). These data suggest that monomeric Ab donkey anti-goat IgG; for all three markers, Alexa Fluor 594 donkey anti- clearance is reduced in HIV-1–infected MDM. rabbit IgG) for 20 min at room temperature. Nuclei were stained with Hoechst 33342 (1:1000). Images were acquired using a Zeiss 510 Meta Confocal Laser Scanning Microscope (Carl Zeiss) at an objective of 340 NEP activity was inhibited by HIV infection of MDM 3 and a 2 zoom view for the interested area. NEP and IDE are two extensively characterized Ab-degrading Statistical tests enzymes, although their enzyme activities in human MDM have not been well characterized. We have compared the activities of Data were presented as means 6 SD unless otherwise noted. All experi- ments were repeated at least three times with different donors, and all data these enzymes in MDM using the fluorogenic peptide Mca- were evaluated statistically by the ANOVA, followed by Newman-Keuls RPPGFSAFK(Dnp)-OH and inhibitors for IDE and NEP (insulin multiple comparison tests using software (Prism 4.0; GraphPad, La Jolla, and thiorphan, respectively). NEP showed ∼4-fold higher endo- CA). In the case of single mean comparison, data were analyzed by t test. p peptidase activity than IDE in MDM at both days 3 and 7 after M- values ,0.05 were regarded as statistically significant. CSF treatment (Fig. 2), suggesting that NEP is a dominant Ab- degrading enzyme during the monocytic differentiation into Results MDM. We next examined the effect of viral infection on NEP b HIV reduced A degradation in MDM activity in MDM at 3, 7, and 10 dpi. NEP endopeptidase activity We have previously shown that Ab degradation was reduced in was significantly decreased by HIV infection by 28.6, 58.4, and HIV-infected MDM as determined by the clearance of aggregated 67.6% at 3, 7, and 10 dpi, respectively (Fig. 3). IDE activity was [125I]-Ab (5). To understand the changes in the clearance of relatively very low and unchanged by viral infection (data not Downloaded from monomeric Ab, we have developed an experimental model for the shown). The infection efficiency was determined by HIV-1 p24 clearance of monomeric Ab after phagocytosis in MDM as de- staining of MDM postinfection (Fig. 4, green), which was 68– scribed previously (45). Briefly, MDM were infected with HIV- 100% (average 89%) at 3 dpi and 100% at 7 and 10 dpi, regardless 1pYu-2 for 24 h, and at 3 dpi, the cells were incubated with 10 mM of donors. monomeric Ab42 for 1 h; 24 h later, cells were then subjected to To understand the mechanism responsible for reduced NEP immunofluorescence of NU-2 for Ab and Hoechst 33342 for activity in HIV-infected MDM, we have examined the protein ex- http://www.jimmunol.org/ nuclear staining (Fig. 1A–H). The intensity of NU-2 staining was pression levels of IDE and NEP by Western blotting. Neither IDE enhanced by viral infection (Fig. 1C,1G), consistent with the nor NEP protein levels were reduced after HIV infection (Fig. 5). by guest on September 26, 2021

FIGURE 7. Laser-scanning confocal microscopy imaging of subcellular 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. Nuclei were visualized with Hoechst 33342 (blue). Images were taken with a confocal microscope at an objective of 340 and 32 zoom view for the ROI. Arrows in- dicate colocalized staining between red and green. 6930 HIV-1 REGULATION OF NEPRILYSIN IN MACROPHAGES

This suggests that the alteration in NEP activity is not due to the changes in its expression level, but is potentially due to its post- translational modifications or alterations in its cellular distribu- tion. Because HIV-Tat protein has been shown to inhibit NEP activity (37), we have examined the direct effect of different doses of intact Tat protein (Tat1–72, 0.1–10 pg/ml) and gp120 (1–100 mM) on the activities of purified recombinant human NEP and IDE proteins in vitro. However, neither Tat nor gp120 protein inhibited NEP or IDE activity, ruling out the potential that these proteins directly inhibit the Ab-degrading enzymes (data not shown). Reduction of cell-surface NEP in HIV-infected MDM NEP activity is sensitive to the changes of pH and has maximum activity at neutral pH, suggesting that its distribution on the cell surface is critical for its maximum activity as compared with its intracellular distribution in acidic environments, such as endo- somes, lysosomes, or ER, where trafficking of extracellular pep- tides are negligible (46). Thus, we have examined if viral infection Downloaded from can alter cellular distribution of NEP and hence its activity. MDM were infected with HIV-1pYu-2 and fixed for immunofluorescence FIGURE 8. HIV-1 infection reduced Ab degradation and NEP activity of NEP under both permeabilized and nonpermeabilized con- in microglia. A, Human microglia (5 3 104 cells/well in 96-well plates) ditions at 7 dpi (Fig. 6). Infection efficiency of the MDM was were infected (HIV group) or uninfected (Control group) with HIV-1pYu2 ∼100% as determined by HIV-1 p24 staining. Using an Ab that for 3 d and tested for Ab42 degradation at 1 and 5 mM concentrations as described in Fig. 1. The cells were subjected to immunofluorescence with

recognizes the extracellular domain of NEP, we revealed that NEP http://www.jimmunol.org/ was prominently expressed on the cell surface in the control group anti-Ab Ab (6E10) and Hoechst 33342 for fluorescent intensity quantifi- (Fig. 6A). However, almost no NEP was detected on the surface of cation of Ab oligomer fluorescent signal (excitation/emission = 488 nm/ 519 nm), which was normalized by the nuclear staining signal (excitation/ MDM after viral infection (90% inhibition versus control, Fig. 6A, emission = 350 nm/461 nm; n = 5 per group). The results were presented as 6B). In contrast, cytoplasmic NEP intensity was stronger in HIV- relative fluorescence ratio. **p , 0.01 by t test. B, Human microglia (1 3 infected MDM when compared with that of the uninfected group 106 cells/well in six-well plates) were infected (HIV group) or uninfected

(247% increase versus control, Fig. 6A–D). In addition, phal- (Control group) with HIV-1pYu2 for 3 d, and cell lysates were collected for loiding staining (red) representing the F-actin signal was consis- NEP activity determination shown as relative fluorescent units. *p , 0.05 tent among MDM with or without infection (Fig. 5B). by Student t test assay.

To further understand the redistribution of NEP after viral in- by guest on September 26, 2021 fection, we performed laser-scanning confocal microscopy to detect the colocalization of NEP with organelle markers. In control duction versus control, Fig. 8B), although the difference was much uninfected MDM, intracellular NEP was mainly colocalized with smaller than the one we saw in virus-infected MDM. This suggests the ER (GRP94), lysosomes (LAMP1), and, to a lesser extent, the that total NEP activity is relatively unchanged and does not di- Golgi apparatus (GM130) (Fig. 6). Viral infection of MDM en- rectly reflect the reduced Ab clearance in virus-infected microglia, hanced the cytoplasmic NEP signal intensity relative to the un- consistent with our finding that the alteration in cellular distri- infected control group, part of which shows colocalization in the bution of NEP plays a role in Ab clearance, as demonstrated in ER and lysosomes (arrows in GRP94 and LAMP1 panels, Fig. 7). MDM. These data suggest that Ab clearance and NEP activity are However, NEP also was found to accumulate in the cytoplasmic also compromised by HIV infection of human microglia. organelles other than either the ER or , presumably endosomes. These results show that HIV infection dramatically restrained the cell surface expression of NEP, resulting in accu- Discussion mulation of cytoplasmic NEP in the ER and lysosomes. This could Understanding the regulation of amyloid clearance in the context of be due to impaired posttranslational modification of NEP at the an HIV infection is critical for the prevention of the early onset of ER or enhanced endocytosis of cell-surface NEP to lysosome due senile dementia in affected populations. Perivascular macrophages to viral infection, leading to NEP inactivation. play an important role in the clearance of Ab exported from the brain, which might be compromised by HIV viral infection. In this b HIV infection inhibited A degradation in microglia present study, the clearance of monomeric Ab is significantly To understand if Ab clearance is compromised by viral infection inhibited by viral infection of MDM, which express both IDE and in the CNS, we have tested if HIV also inhibits degradation of NEP as Ab-degrading enzymes. NEP activity is more potent than monomeric Ab in microglia. For that purpose, primary cultured IDE activity in uninfected, differentiated MDM, and the viral human microglia were infected with HIV-1pYu-2 for 3 d, followed infection significantly compromises the enzyme activity as de- by incubation with different doses of monomeric Ab and immu- termined by the virus-infected MDM lysates. Although the viral nofluorescence with anti-Ab Ab 6E10 after 24 h incubation. infection does not alter the cellular protein expression of NEP or Consistent with the result found in MDM, HIV infection of IDE, it greatly reduces the cell-surface NEP level. We have shown microglia significantly increased the fluorescent intensity of Ab at that HIV infection induces an intracellular redistribution of NEP both 1 and 5 mM doses (55 and 132% increase versus control, primarily to the ER and lysosomal compartments. An additional, respectively), suggesting that reduced Ab clearance occurs in intracellular compartment of NEP may also include endosomesal primary microglia as well (Fig. 8A). In accord, NEP activity was compartments considering an internalization mechanism of the also significantly reduced by viral infection at 3 dpi (12% re- enzyme. The Journal of Immunology 6931

Because NEP is a neutral zinc-dependent metalloprotease and its In addition, HIV viral infection may also compromise activity of activity is diminished at lower pH or in the absence of zinc (46), NEP and IDE by virtue of deregulating apolipoprotein E (ApoE), ER, lysosomal, or endosomeal-localized NEP might not have which plays a role in facilitating the proteolytic clearance of soluble optimal endopeptidase activity. Therefore, HIV infection leading Ab from the brain by NEP and IDE (53). It was reported that in to a redistribution of NEP to intracellular compartments with HIV-infected brains, binding of viral Tat protein to low-density lower than normal physiologically pH is likely to impair NEP lipoprotein receptor-related protein resulted in substantial in- enzyme activity. Indeed, redistribution of NEP to specific in- hibition of neuronal binding, uptake, and degradation of physio- tracellular compartments by fusing recombinant NEP to different logical ligands for low-density lipoprotein receptor-related protein organelle-specific molecules results in diminished NEP activity; such as ApoE (54). Hence, it would be beneficial to examine if the wild-type NEP has the most enzyme activity, whereas the NEP there is an alteration of ApoE levels in HIV-infected brains. Ad- fused to ER-targeting molecule demonstrated the least activity ditionally, brain ApoE expression may be reduced by HIV in- (47). Indeed, it has been reported that PBMCs from HIV-1– fection because it has been shown that HIV downregulates renal infected subjects have abnormal cell-surface enzyme kinetics ApoE expression (55). (including NEP), and the subcellular distribution of these enzymes Interestingly, antioxidant or anti-inflammatory supplements was also markedly changed (48). Thus, it is possible that im- could potentially alleviate the oxidation-mediated impairment of pairment of Ab clearance at early time points may be attributed to NEP activity and enhance Ab clearance in HIV-affected brains. the reduction in cell-surface level of NEP (where NEP is situated Indeed, it has previously been shown that simavastatin, an anti- in optimal pH). inflammatory compound, can attenuate virus-induced Ab accu-

The mechanism of virus-induced redistribution of NEP is un- mulation in brain endothelial cells (56). Taken together, restora- Downloaded from known. One possibility is the endocytosis of NEP along with viral tion of NEP activity in the brain at the early stages of HIV- infection of MDM. However, so far, there is no specific viral capsid associated dementia may represent an effective strategy to pre- or envelope protein reported to interact with NEP for the induction vent or attenuate disease progression. of endocytosis. HIV gp120 had no effect on either purified NEP or Finally, it should be noted that viral infection of mononuclear IDE activity in vitro. Alternatively, NEP may be endocytosed along phagocytes induces innate immunity responses, which are char-

with viral endocytosis as a bystander membrane protein, effectively acteristic proinflammatory activation. This leads to enhanced http://www.jimmunol.org/ inducing the imbalance of cell-surface NEP and recycling/ production of proinflammatory cytokines, such as IFN-g and TNF- endocytosed NEP after viral infection. HIV-1 Tat can bind to a, which enhances production of Ab through upregulation of APP NEP and inhibit its enzyme activity in primary cultured neurons and b-site APP-converting enzyme 1 from neurons and astrocytes and purified enzyme systems (37, 38). However, intact Tat protein (23). Proinflammatory cytokines also potently suppress Ab clear- failed to inhibit purified recombinant NEP in our system. Al- ance from human mononuclear phagocytes via downregulation though there is a possibility that Tat protein is partially processed of Ab-degrading enzymes and chaperone molecules involved in in the cells and has NEP inhibitory activity in MDM, these data protein refolding (39). Thus, bystander effect of brain parenchyma suggest that the reduced NEP activity by HIV infection is not due activation should be taken into account for understanding the to the direct effect of viral proteins, but could be due to their overall mechanism of b-amyloidosis in brain. by guest on September 26, 2021 interaction with NEP for its endocytosis or impairment of post- In summary, we demonstrate that monomeric Ab degradation is translational modification of NEP as part of viral-suppression reduced by HIV infection of human MDM as early as 3 dpi. This mechanisms. is accompanied by redistribution of cell-surface NEP to intra- Some posttranslational modification of NEP can also be altered cellular compartments at 3 dpi and reduction in NEP activity in by viral infection. An alternative mechanism affecting NEP surface cell lysates at 7 dpi without alteration of cellular expression levels expression is glycosylation, which plays a critical role in regulating of NEP or IDE. Viral Tat or pg120 has no effect on purified IDE or transport of NEP to the cell surface as well as the enzymatic activity NEP enzyme activity in vitro. Comparable results are also found of NEP (46). Decreased glycosylation results in reduced transport in human primary cultured microglia, suggesting that viral in- of NEP to the cell surface; thus, HIV infection may somehow fection also compromises microglial Ab clearance in the brain. inhibit the glycosylation level of NEP. Unfortunately, the large These data suggest that modulation of amyloid clearance by pe- bands of the NEP on the immunoblotting data do not allow us to ripheral macrophages is a potential preventative target of Ab-re- observe m.w. modifications, precluding from the conclusion. lated cognitive disorders in affected populations. Another possibility lies in that HIV infection enhances endocy- tosis by viral infection and entry, and this may then lead to the Acknowledgments enhanced relocation of NEP into endosomes (49). However, the We thank Kathy Estes, Kaitlin Ingraham, and Rob Freilich for manuscript detailed mechanisms responsible for disrupting NEP translocation editing, William Klein for NU-1 Ab, and Li Wu for tissue culture. from the plasma membrane by HIV infection remain to be elu- cidated in future studies. Disclosures One caveat of this NEP redistribution theory is that it does not The authors have no financial conflicts of interest. explain the mechanism of reduced NEP activity in the virus- infected MDM lysate, which is assayed at an optimum pH con- References dition (pH 7.5). HIV-infected cells are in a cellular redox state, 1. Gissle´n, M., J. Krut, U. Andreasson, K. Blennow, P. Cinque, B. J. Brew, which is closely linked to an increased level of ceramide, sphin- S. Spudich, L. Hagberg, L. Rosengren, R. W. Price, and H. Zetterberg. 2009. Amyloid and tau cerebrospinal fluid biomarkers in HIV infection. BMC Neurol. gomyelin, and 4-hydroxynonenal. These oxidative stress markers 9: 63. were increased in HIVencephalitis (48, 49). It was shown that NEP 2. Valcour, V. G., C. M. Shikuma, M. R. Watters, and N. C. Sacktor. 2004. Cog- nitive impairment in older HIV-1-seropositive individuals: prevalence and po- is modified by 4-hydroxynonenal adducts, resulting in decreased tential mechanisms. AIDS 18(Suppl 1): S79–S86. activity in the brain of AD patients and cultured cells (50–52). 3. Pulliam, L. 2009. HIV regulation of amyloid beta production. J. Neuroimmune Pharmacol. 4: 213–217. Thus, IDE and NEP may undergo oxidation-mediated modifi- 4. Esiri, M. M., S. C. Biddolph, and C. S. Morris. 1998. Prevalence of Alzheimer cations after viral infection and are therefore inactivated. plaques in AIDS. J. Neurol. Neurosurg. Psychiatry 65: 29–33. 6932 HIV-1 REGULATION OF NEPRILYSIN IN MACROPHAGES

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