nef-deleted HIV-1 inhibits phagocytosis by monocyte-derived macrophages in vitro but not by peripheral blood monocytes in vivo
Kedzierska, Katherinea,f; Mak, Johnsona,g,*; Jaworowski, Anthonya,e,*; Greenway, Alisonb,g,*; Violo, Antoniettea; Chan, HiuTata; Hocking, Janec; Purcell, Damianb; Sullivan, John S.h; Mills, Johnd,e; Crowe, Suzannea,e,f
From the aAIDS Pathogenesis Research Unit, the bAIDS Cellular Biology Unit and the cEpidemiology and Social Research Unit, the dChildren's Virology Research Unit, Macfarlane Burnet Centre, the eNational Centre for HIV Virology Research, the fDepartment of Medicine, Monash University, Melbourne, the gDepartment of Biochemistry, Monash University, Melbourne, Australia, and the hAustralian Red Cross Blood Service, Sydney Australia. Note: *These authors contributed equally to the research.
Correspondence to Professor Suzanne M Crowe, AIDS Pathogenesis Research Unit, Macfarlane Burnet Centre, Yarra Bend Road, Fairfield, Victoria, Australia 3078. Tel: +61 3 9282 2194; fax: +61 3 9482 6152; email: email@example.com
Received: 17 November 2000;
revised: 8 February 2001; accepted: 14 February 2001.
Sponsorship: This work was supported by funding from the National Centre in HIV Virology Research and the Macfarlane Burnet Centre Research Fund. K.K. is a recipient of a National Health and Medical Research Council Postgraduate Scholarship.
Objective: HIV-1 infection impairs a number of macrophage effector functions, but the mechanism is unknown. We studied the role of HIV-1 Nef in modulating phagocytosis by human monocytes and monocyte-derived macrophages (MDM).
Design and methods: Using a flow cytometric assay, phagocytosis of Mycobacterium avium complex (MAC) by monocytes in whole blood of Sydney Blood Bank Cohort (SBBC) members infected with a nef-deleted (Δnef) strain of HIV-1 was compared with that of monocytes from uninfected or wild-type (WT) HIV-infected subjects. The specific impact of Nef on phagocytosis by MDM was determined by either infecting cells in vitro with Δnef strains of HIV-1 or electroporating Nef into uninfected MDM.
Results: MAC phagocytic capacity of monocytes from SBBC members was equivalent to that of cells from uninfected individuals (P = 0.81); it was greater than that of cells from individuals infected with WT HIV-1 (P < 0.0001), irrespective of CD4 counts and HIV viral load. In contrast, in vitro infection of MDM with either Δnef or WT strains of HIV-1 resulted in similar levels of HIV replication and equivalent impairment of phagocytosis via Fcγ and complement receptors. Electroporation of Nef into MDM did not alter phagocytic capacity.
Conclusions: This study provides evidence demonstrating the complex indirect effect of Nef on phagocytosis by peripheral blood monocytes (infrequently infected with HIV-1) in vivo. Conversely, the fact that MDM infected with either Δnef or WT HIV-1 in vitro (high multiplicity of infection) show comparably impaired phagocytosis, indicates that HIV-1 infection of macrophages can directly impair function, independent of Nef.
Cells of macrophage lineage including peripheral blood monocytes and tissue macrophages are important effector cells against a number of intracellular pathogens including Mycobacterium avium complex (MAC), Toxoplasma gondii, Candida albicans and Pneumocystis carinii. These cells provide critical functions in the cell-mediated response to these opportunistic pathogens such as chemotaxis, phagocytosis and intracellular killing. We and others have reported that a number of these functions are impaired following HIV-1 infection [1–3]. These defects contribute to the pathogenesis of AIDS by allowing reactivation or infection with otherwise uncommon opportunistic pathogens (reviewed in ), resulting in significant morbidity and mortality.
The exact mechanism by which HIV-1 impairs monocyte/macrophage function and promotes disease progression remains unclear. However, the importance of Nef (a 25 to 30 kDa myristylated accessory protein) for HIV-1 pathogenesis has been demonstrated in prospective studies of the Sydney Blood Bank Cohort (SBBC), a blood donor and eight transfusion recipients who were all infected with a strain of HIV-1 containing deletions within the nef gene and deletions and duplications within the overlapping long terminal repeat overlap [5–7]. Although there is evidence of recent clinical progression in two of the cohort members after 16 years of infection, the virus is clearly attenuated in comparison with wild-type (WT) HIV-1 strains, supporting the role for a functional Nef in HIV-1 pathogenesis in humans [5–7]. An intact nef gene has also been shown to be important for disease progression in simian immunodeficiency virus (SIV)-infected adult rhesus macaques  as well as in HIV-infected severe combined immunodeficient (SCID)-hu mice . However, viral load may play an important role, as infant macaques infected with nef-deleted SIV at high multiplicity of infection developed disease .
Nef is thought to contribute to HIV-1 pathogenesis through a variety of mechanisms such as down-regulating CD4 and MHC class I expression [11–15], enhancing virion infectivity [16–18] and modulating signalling pathways via interactions with host cell proteins (reviewed in ). As these latter include interactions of Nef with cellular proteins and kinases which are also involved in the process of phagocytosis (such as Src kinases, Hck and Lyn [20–23], p21-activated kinase [24,25], guanine-nucleotide exchange factor Vav  and small GTPases Rac1 and Cdc42 [27,28]), it is possible that Nef modulation of signalling pathways involving these proteins inhibits phagocytosis. The role of Nef on phagocytosis by human monocytes and macrophages following HIV-1 infection has not yet been elucidated.
In this study we have investigated the effects of Nef on phagocytosis of MAC, by comparing monocytes from members of SBBC with monocytes from subjects who are either uninfected or infected with WT HIV-1. We have examined the indirect (e.g. cytokine or chemokine) effects of HIV-1 infection on phagocytosis by monocytes (infrequently infected with HIV-1) in whole blood from HIV-infected individuals including SBBC members and patients with WT HIV-1 infection. To examine the direct effects of HIV-1 replication within monocyte-derived macrophages (MDM) we have infected MDM with either nef-deleted (Δnef) or WT strains of HIV-1 in vitro, at relatively high multiplicity of infection, and assessed phagocytosis of both IgG- and complement (C′)-opsonized targets. To examine the effects of Nef on phagocytosis directly we have electroporated Nef protein into uninfected MDM. The results of this study demonstrate that the impact of HIV-1 Nef protein on phagocytosis differs markedly in MDM infected in vitro and in monocytes studied ex vivo, reflecting the potentially different mechanisms underlying inhibition of phagocytosis in these two situations.
Sources of monocytes
Blood (2 ml) was collected from six members of the Sydney Blood Bank Cohort in lithium heparin anticoagulant, with their informed consent, and dispensed into polypropylene tubes. Similarly, blood was collected from HIV-1-infected patients and from uninfected controls. Peripheral blood was assessed for plasma HIV RNA by bDNA assay (Chiron Corporation, Emeryville, USA) or reverse transcriptase (RT)-polymerase chain reaction (Roche Diagnostics, Nutley, New Jersey, USA) according to manufacturers’ instructions.
Whole blood phagocytosis assay
Fluorescein isothiocyanate (FITC)-labelled MAC (5 × 106 and 1.5 × 107) was added to 100 μl of blood dispensed in polypropylene tubes (Becton Dickinson, Franklin Lakes, New Jersey, USA) and placed into a shaking waterbath at 37°C for phagocytosis to proceed, as previously described by this laboratory [3,29]. Phagocytosis was terminated after 10 min by plunging the tubes into ice. The fluorescence of MAC-FITC adherent to the monocyte surface was quenched using quenching agent (Orpegen, Heidelberg, Germany). Monocytes were identified by staining with anti-CD14 conjugated to phycoerythrin (Becton Dickinson). Erythrocytes were lysed with FACS lysing solution (Becton Dickinson) and the remaining cells were fixed with 1% formaldehyde (Polysciences, Warrington, Pennsylvania, USA) for flow cytometric analysis (FACStarPLUS). The proportion of monocytes that had ingested MAC-FITC were plotted against the ratio of MAC-FITC : monocyte which was calculated from the total monocyte count as previously described .
F-actin content of peripheral blood monocytes during phagocytosis
Blood (100 μl) was dispensed in polypropylene tubes and cooled on ice for 20 min. To perform phagocytosis, blood samples were incubated with or without IgG-opsonized latex beads (3 μm in diameter; Sigma, St Louis, Missouri, USA) at a concentration of 5 × 107 beads/ml, at 37°C. Phagocytosis was terminated at various times (0 to 10 min) by plunging the tubes into ice and fixing the cells with 1 ml of 3% formaldehyde (20 min, 4°C). Following two washes with cold 0.1 mol/l glycine in phosphate-buffered saline (PBS)-calcium magnesium free (CMF), the monocytes within blood were stained with anti-CD14 Mab conjugated to phycoerythrin for 30 mins at 4°C. After a wash in cold (4°C) PBS-CMF, the erythrocytes were lysed with FACS lysing solution at 4°C. The remaining white blood cells were permeabilized with 0.1% Triton-X 100 (Merck, Kilsyth, Australia) for 1 min, washed twice with 1% FCS/PBS-CMF and stained for F-actin levels with phalloidin-Alexa 488 (Molecular Probes, Eugene, Oregon, USA) for 30 min at 4°C. The cells were washed with cold PBS-CMF, fixed with 1% formaldehyde and analysed by flow cytometry.
HIV-1 infection of MDM in vitro
A subproviral Δnef construct, obtained from NIH AIDS Reagent Program (contributed by Ronald Desrosiers), was used to generate the full length NL4.3Δnef proviral DNA. The DNA constructs pNL(AD8) and pNL(AD8Δnef) were prepared by substituting the respective envelope coding DNA sequences from NL4.3 and NL4.3Δnef with monocytotropic AD8 envelope coding sequences, converting a T-tropic virus to M-tropic. A laboratory-adapted M-tropic strain (HIV-1Ba−L) of HIV-1 was also used for MDM infections as described previously . The nef-deleted primary isolates from D36 and C18 (two of the SBBC members from which virus could be successfully isolated) were prepared by co-culturing peripheral blood mononuclear cells (PBMC) from D36 or C18 with HIV-1 seronegative CD8-depleted PBMC that were pre-stimulated with phytohaemagglutinin (10 μg/ml; Murex Diagnostics, Dartford, UK) and macrophage colony-stimulating factor (750U/ml, Genzyme, Cambridge, Massachusetts, USA) for 3 days prior to co-culture. Cells were then co-cultured in the presence of recombinant human interleukin (IL)-2 (10 U/ml; Boehringer Mannheim, Mannheim, Germany). Purified MDMs from HIV-1 seronegative buffy coats were infected with HIV-1NL(AD8) chimera, HIV-1 NL(AD8Δnef) chimera, HIV-1Ba−L, and primary isolates HIV-1D36 or HIV-1C18 from SBBC members at the same multiplicity of infection. Control cells were mock-infected and cultured under identical conditions. HIV-1 infections were all performed using MDM 5 days after their isolation, cultured in suspension in polytetrafluorethylene (Teflon) jars (Savillex, Minnetonka, Minnesota, USA) at a concentration of 1 × 106 cells/ml. HIV-1 replication in MDM was quantified by flow cytometry by measuring intracellular p24 antigen using a MAb directed against p24 (2μg/ml; IgG1, Olympus, Lake Success, New York, USA), followed by goat anti-mouse IgG conjugated to FITC (FITC-GAM; Tago, Burlingame, California, USA) or by monitoring RT activity using a micro RT assay as previously described .
Phagocytosis by MDM infected with HIV-1 in vitro
Phagocytosis of MAC-FITC
The phagocytic capacity of MDM for MAC-FITC was assessed 7 days after HIV-1 infection. Cells (2 × 105 cells in 100 μl of PBS) were dispensed in polypropylene tubes (Becton Dickinson) and cooled on ice for 20 min. MDM were incubated at 37°C (or on ice as a control) for 2 h with MAC-FITC, at various MAC-FITC : MDM ratios (ranged from 1 : 10 to 1 : 75), in duplicate. After washing MDM with PBS and quenching adherent MAC-FITC, the cells were fixed and the proportion of MDM that had ingested MAC-FITC was quantified by flow cytometry as previously described .
Fc-gamma receptor (FcγR)-mediated phagocytosis
On day 7–10 following HIV-1 infection, the MDM were plated onto 96-well plates (Costar, Cambridge, Massachusetts, USA) at 5 × 104 cells per well in 100 μl of supplemented Iscove's medium (Cytosystem, Castle Hill, Australia), and allowed to adhere for 2 h in a 37°C, 5%CO2 humidified incubator. Sheep red blood cells (sRBC; ICN-Cappel, Aurora, Ohio, USA) were opsonized immediately prior to the phagocytosis assay with a subagglutinating titre of rabbit anti-sRBC antibody (1 : 300; ICN-Cappel) for 30 min at room temperature. IgG-opsonized or unopsonized sRBC were added to adhered MDM at a sRBC : MDM ratio of 10 : 1. The plate was centrifuged at 100 ×g for 5 min at 4°C and then placed at 37°C, 5%CO2 for phagocytosis to proceed. Phagocytosis was terminated after 10 min by placing the plates on ice and washing the cells with ice-cold PBS. The level of phagocytosed sRBC was determined by colorimetric assay . Briefly, after unbound sRBC were removed by washing with PBS, the bound non-phagocytosed sRBC were lysed with 0.2% NaCl for 3 min. Phagocytosed sRBC were assessed after total cell lysis in 0.2 mol/l Tris-HCl buffer containing 6 mol/l urea by reaction of haemoglobin with 2,7-diaminoflurene (Sigma). Absorbance was determined at 620 nm in a plate reader (Labsystems, Multiskan, Helsinki, Finland), and compared with a standard curve generated using known amounts of sRBCs (ranging from 2 × 103 to 5 × 105).
sRBC were opsonized with pre-warmed 2% AB-negative human serum as a source of human complement components for 30 min at room temperature (Chan H-T, unpublished). As controls, sRBC were opsonized with the same source of AB-negative serum that had been heat-inactivated at 56°C for 45 min (HI-sRBC). Prior to the phagocytosis assay, MDM that had adhered to 96-well plates were activated by treatment with phorbol-12-myristate-13-acetate (PMA) at a final concentration of 200 nmol/l per well for 10 min at 37°C. Subsequently those cells were exposed to C′-sRBC or HI-sRBC at an sRBC : MDM ratio of 20 : 1. C′R-mediated phagocytosis was performed at 37°C for 60 min and assessed by colorimetric assay as described above for FcγR-mediated phagocytosis.
Phagocytosis by Nef-electroporated MDM
To assess the direct effect of Nef on phagocytosis in vitro, purified recombinant Nef protein  was introduced by electroporation into MDM on day 5 post-isolation. Cells that were electroporated with purified recombinant glutathione-s-transferase (GST) or mock-electroporated (no protein) were used as controls. Proteins (300 nmol/l per 1 × 106 cells) were introduced into MDM using a square-wave electroporator (Bio-Rad, Hercules, California, USA; amplitude, 5 kV; pulse frequency, 28; burst time, 0.8 s; cycle number, 10). Electroporation of Nef into MDM was confirmed by immunofluorescence staining using specific anti-Nef MAb (AE6; AIDS Research and Reference Reagent Program, NIAID, NIH: HIV-1 Nef monoclonal antibody from Dr James Hoxie) immediately after electroporation and at later times as previously described . Electroporated MDM were returned to cultures and allowed to recover for 24 h at 37°C in a humidified incubator and subsequently used for phagocytosis assays. The impact of Nef on macrophage function was investigated by assessing its role on MAC-FITC phagocytosis (as described above) as well as on the level of tyrosine phosphorylation during FcγR-mediated phagocytosis. The latter was performed by incubating MDM with IgG-opsonized latex beads at a ratio of 1 : 10 respectively at 37°C. At indicated time points phagocytosis was arrested by plunging the tubes into ice and washing MDM in ice-cold PBS, followed by centrifugation at 14 000 rpm for 45 s. Cells were lysed in 100 μl of Triton-X lysis buffer containing 25 mmol/l Tris-HCl (pH 7.5), 0.14 mol/l NaCl, 1 mmol/l EDTA, 1% Triton-X-100, supplemented with the following phosphatase inhibitors: 50 mmol/l NaF, 1 mmol/l sodium orthovanadate (Sigma), 40mmol/l β-glycerophosphate (Sigma), and the following protease inhibitors: 1 mmol/l pefabloc, 1 μmol/l pepstatin, 1 μmol/l leupeptin (Boehringer-Mannheim). After 30 min lysis at 4°C, MDM extracts were centrifuged at 14 000 rpm for 10 min and assessed by immunoblot analysis.
Cell extracts containing equal amounts of proteins, as determined by DC protein assay (Bio-Rad Laboratories), were boiled in sodium dodecyl sulphate (SDS) sample buffer (10 mmol/l Tris pH 8.0, 2 mmol/l EDTA, 1% SDS, 5% β-mercaptoethanol, 5% glycerol) resolved by 10% SDS-polyacrylamide gel (SDS-PAGE), transferred to Hybond-C nitrocellulose (Amersham Pharmacia, Little Chalfont, Buckinghamshire, UK) and blocked for 2 h in 3% bovine serum albumin (Sigma). The blots were probed with a recombinant antibody directed against phosphotyrosine conjugated with horseradish peroxidase (RC20; Transduction Laboratories, Lexington, Kentucky, USA) overnight at 4°C, washed five times in 1 × Tris-buffered saline containing 0.3% Tween-20 (Astral, Gymea, Australia) and subsequently developed for enhanced chemiluminescence (ECL) according to manufacturer's instructions (Amersham Pharmacia).
Multiple regression was used to model the association between the percentage of phagocytosis and MAC-FITC:monocyte ratio, adjusting for HIV-1 infection status (HIV-1 positive, HIV-1 negative, and nef-deleted SBBC HIV-1 positive). Robust standard errors were used to account for repeat tests on individuals. To account for the highly skewed data, the MAC-FITC : monocyte ratio was transformed using natural logarithms. Interactions between MAC-FITC : monocyte ratio and HIV infection status were investigated by fitting interaction terms in the model. The analyses were carried out using Stata statistical software analysis package (Stata, Collage Station, Texas, USA).
The significance of F-actin contents of monocytes from HIV-positive and -negative individuals, in vitro infections with WT HIV-1(HIV-1Ba-L or HIV-1NL (AD8)) and nef-deleted strains of HIV-1 (primary isolates HIV-1D36 and HIV-1C18 or HIV-1NL (AD8)(nef chimera) as well as the impact of Nef electroporation on phagocytosis was assessed using the Student's t test (paired, two-tailed).
Blood was collected from a total of six Sydney Blood Bank Cohort members (median CD4, 805 × 106 cells/μl; range, 306 to 2331 × 106 cells/μl; median plasma HIV RNA < 400 copies/ml; range, < 400 to 4000 copies/ml), 16 persons with WT HIV-1 infection (median CD4, 138 cells × 106 cells/μl; range, 20 to 713 × 106 cells/μl for 11 subjects tested at the time of assay; median plasma HIV RNA 900 copies/ml; range, < 500 to 385 500 copies/ml for 13 subjects tested) and three uninfected persons. Blood samples from the same HIV-uninfected donors were used as a source of control monocytes in every experiment (Table 1).
Phagocytosis by monocytes in whole blood from individuals infected with WT and nef-deleted HIV-1
As SBBC members have been infected with an attenuated strain of HIV-1 for periods in excess of 16 years prior to the onset of any HIV-related symptoms or infections , it was of interest to determine whether they had unimpaired phagocytic function, consistent with slow or absent disease progression. Monocytes present in whole blood from members of SBBC phagocytozed MAC-FITC (ingested predominantly via C′R ) with an efficiency similar to that of monocytes from HIV-1-uninfected individuals (P = 0.808), but significantly better than monocytes from individuals infected with WT HIV-1 (P < 0.0001) (Table 1, Fig. 1). In addition, monocytes from members of SBBC phagocytosed Toxoplasma gondii–FITC (mediated predominantly via FcγRII ) substantially better than monocytes from WT HIV-infected individuals (n = 4; data not shown). These data suggest that both C′R- and FcγR-mediated phagocytosis by monocytes from WT HIV-infected individuals is impaired, whereas those from subjects infected with Δnef HIV-1 are not. No correlation was found between the level of inhibition of phagocytosis and either viral load (R = −0.27;P = 0.38) or CD4 counts (R = 0.35;P = 0.29).
Mechanism underlying inhibition of phagocytosis
The surface expression of the complement receptor (CD11c) and Fcγ receptors (CD16, CD32) was not modulated in monocytes from individuals infected with WT or nef-deleted HIV-1 when compared with uninfected subjects (data not shown), suggesting a post-receptor mediated mechanism of phagocytosis inhibition in individuals infected with WT HIV-1. As reorganization of the actin-based cytoskeleton is essential for the formation of the phagocytic cup and the engulfment of phagocytosed particles, the mechanism of defective phagocytosis in HIV-infected individuals was further investigated at the actin polymerization level. A flow cytometric assay was developed to measure the level of polymerized actin (filamentous actin; F-actin) in blood monocytes during FcγR-mediated phagocytosis (Kedzierska, unpublished). Monocytes in blood of HIV-infected individuals showed significantly increased basal levels of polymerized actin in comparison with F-actin level in monocytes from uninfected controls (Fig. 2a;P < 0.05; n = 5). During phagocytosis there was a significant net increase of F-actin from basal levels in monocytes from uninfected controls at between 2 and 10 min of phagocytosis, but not in monocytes from HIV-infected individuals (Fig. 2b; n = 3). These data suggest defective actin rearrangement (and thus abnormal phagocytic cup formation) in blood monocytes from HIV-infected subjects.
Phagocytosis by purified MDM infected with HIV-1 in vitro
As phagocytosis by monocytes from SBBC members was not impaired, the direct effect of Nef on phagocytosis was assessed, initially by using Δnef strains to infect MDM in vitro in comparison with WT HIV-1. In vitro infection of MDM by Δnef strains HIV-1D36 (primary isolate from SBBC member D36) or HIV-1NL(AD8)Dnef chimera impaired phagocytosis of MAC-FITC to the same degree as infection with WT HIV-1 [Ba-L or NL(AD8)]. All infected MDM cultures showed impaired phagocytosis compared with uninfected MDM (P < 0.001;Fig. 3).
MDM infected with either WT HIV-1 (Ba-L or NL(AD8)) or Δnef HIV-1C18 (primary isolate from SBBC cohort member C18) or HIV-1NL(AD8)Δnef chimera had impaired phagocytosis mediated via either Fcγ and C′ receptors using specifically opsonized targets (IgG- and C′-opsonized sRBC) in comparison with uninfected MDM (P < 0.001;Fig. 4). Infection of MDM with nef-deleted strains of HIV-1 resulted in similar levels of inhibition of phagocytosis as MDM infected with WT HIV-1 (P = 0.08 and P = 0.1, for FcγR- and C′R-mediated phagocytosis respectively). The presence of a nef-deletion had no significant effect on HIV-1 replication in vitro as measured by RT activity in the culture supernatant (mean RT values of 204 500 cpm/1 × 106 MDM and 125 000 cpm/1 × 106 MDM for HIV-1NL(AD8) and HIV-1NL(AD8)Δnef chimera respectively; n = 5;P = 0.48) or by flow cytometric analysis of intracellular p24 antigen (mean of 59 and 74% p24-positive MDM for HIV-1NL(AD8) and HIV-1NL(AD8)Δnef chimera respectively; n = 2).
Phagocytosis by MDM electroporated with Nef protein
To address directly the effects of Nef on phagocytosis by MDM, cells were electroporated with Nef. Electroporation of Nef protein into MDM did not affect phagocytosis of MAC-FITC compared with mock- or GST-electroporated controls (n = 4;P = 0.16 and 0.54 respectively) (Fig. 5a). As Nef interacts with cellular proteins and kinases which are also phosphorylated during FcγR-mediated phagocytosis (eg Hck), we assessed the impact of Nef on the level of tyrosine phosphorylation during phagocytosis of IgG-opsonized targets. Stimulation of mock-electroporated MDM with IgG-opsonized beads triggered an increase in tyrosine phosphorylation of a wide range of cellular proteins after 2 min of phagocytosis. Nef- and GST-electroporated MDM displayed similar levels and patterns of phosphorylation during FcγR-mediated phagocytosis to that induced by mock electroporation (Fig. 5b). These data suggest that HIV-1 Nef does not inhibit phagocytosis via FcγR or C′R in MDM infected in vitro.
Our data show that phagocytosis of MAC by blood monocytes from individuals infected with nef-deleted strains of HIV-1 was normal, whereas in agreement with our previous data  phagocytosis by blood monocytes from WT HIV-1 infected subjects was significantly impaired, in comparison with monocytes from uninfected controls. However, when MDM from HIV-1 seronegative individuals were infected in vitro at the same multiplicity of infection with WT or Δnef HIV-1 strains, the phagocytic capacity of these MDM were equally reduced. These results suggest that if HIV-1 Nef protein has an effect on monocyte and macrophage phagocytic function, it is mediated through a complex indirect effect only evident in vivo.
A number of investigators have reported impairment of phagocytosis following WT HIV-1 infection, by monocytes and alveolar macrophages in vivo as well as by macrophages infected with WT HIV-1 in vitro[35–42], although others have shown normal monocyte and macrophage function following HIV-1 infection [43,44]. These discordant results may be at least partially explained by the differing methods used by investigators, in particular whether cells have been isolated from HIV-infected individuals (where the proportion of infected cells is very low, in the range of 0.001 to 1%) or from seronegative donors whose purified MDM were then infected in vitro (with infection rates often in the range of 30 to 70%). As previously demonstrated by our group, monocytes and macrophages are distinct cell populations that differ in their susceptibility to HIV-1 infection (monocytes being highly refractory, whereas macrophages are fully permissive to HIV-1) , the expression level of surface receptors (including CD4, CCR5 and phagocytic receptors) [30,46] as well as in their cytokine/chemokine production profile (reviewed in ). This study, however, demonstrates defective phagocytosis by either monocytes or macrophages following HIV-1 infection in vivo and in vitro.
Phagocytosis of opportunistic pathogens such as MAC (utilizing predominantly C′R for phagocytosis;) and Toxoplasma gondii (utilizing FcγRII;) was impaired by peripheral blood monocytes from WT HIV-infected individuals but not from SBBC members infected with a Δnef strain of HIV-1. Impaired phagocytosis following HIV-1 infection is likely to directly contribute to HIV pathogenesis, by allowing reactivation of opportunistic pathogens normally controlled by macrophages. Our data demonstrating normal phagocytic efficiency of monocytes in the blood of SBBC members is therefore consistent with their long-term non/slow progression and the absence of opportunistic infections . As individuals infected with nef-deleted HIV-1 displayed normal phagocytic efficiency, we considered that Nef might be responsible for the observed impairment of phagocytosis following HIV-1 infection. However, as the attenuated virus from this group also has deletions and rearrangements within the long terminal repeat , a tight correlation of phagocytic efficiency and nef-deletion is not possible.
As only a small proportion of blood monocytes (< 1%) is infected with HIV-1, impaired phagocytosis may be predominantly an indirect consequence of HIV-1 infection and might reflect dysregulation of cytokine/chemokine production by monocytes and/or other cells present in blood. In support of this proposal, defective phagocytosis by neutrophils (not targets for HIV-1 infection) from HIV-positive individuals has also been reported . Nef may alter phagocytosis through its influence on the cellular environment and subsequently on cytokine and chemokine production. Nef induces the production of two CC-chemokines, MIP1-α and MIP1-β by macrophages , induces synthesis of IL-15 by MDM , IL-10 , tumour necrosis factor-α, IL-6 and IL-1b (Greenway, unpublished) by PBMC or alters IL-2 production by T lymphocytes [51,52]. As monocyte activation and their response to phagocytic stimuli are dependent on cytokine concentration (including IL-2 ) Nef may impair phagocytosis via dysregulated cytokine/chemokine production.
The nef gene of primate lentiviruses is necessary for high level virus replication in vivo. It has been also demonstrated by the work of Ruprecht et al. that SIV with nef deletion is generally attenuated in adult macaques, but causes disease in infant macaques, probably as a result of a ‘threshold’ for pathogenicity relating to levels of viral replication and viral load [10,54]. Although it is possible that Nef may influence phagocytic capacity of monocytes in vivo by affecting viral load and CD4 counts, our data suggest that the reduction of phagocytosis was not due to viral load or CD4 counts. SBBC members who contributed to this study had low viral load and normal CD4 counts at the time of phagocytosis measurements, whereas individuals infected with WT HIV-1 had a wide range of viral loads and CD4 counts. However, we found no correlation between the observed inhibition of phagocytosis and either viral load (undetectable or low in approximately 50% of subjects) or CD4 counts in individuals infected with WT HIV-1, suggesting that the difference in phagocytosis between nef- deleted and WT HIV-infected individuals is not due to either of those factors. Those results are in the agreement with studies showing decreased phagocytosis in patients with both undetectable (< 400 copies/ml) and high viral loads (> 10 000 copies/ml)  as well as over a wide range of CD4 counts .
The mechanism of defective phagocytosis by peripheral blood monocytes from WT HIV-infected individuals remains unclear. Previous reports [37,38,55], confirmed by our observations, suggest that HIV-1 infection in vivo results in either elevated or unchanged expression of complement and Fcγ receptors on monocytes, indicating that inhibition of phagocytosis occurs at a post-receptor level. Recently, Elbim et al.  showed increased basal levels of F-actin in monocytes from HIV-infected individuals, at different stages of disease, when compared with uninfected subjects. We have confirmed these findings and have further demonstrated that monocytes from HIV-infected individuals display either minimal or no elevation of F-actin above their basal level during phagocytosis. This is in contrast to significant net increases from the basal F-actin level in uninfected controls. As actin polymerization plays a critical role in the formation of phagocytic cup and ingestion of phagocytosed particles, defective actin rearrangement is a potential mechanism underlying impaired phagocytic function in HIV-infected individuals. F-actin content in monocytes from individuals infected with nef-deleted HIV-1 remains to be elucidated.
There are other potential mechanisms by which Nef could directly influence phagocytosis. Nef associates with several cellular kinases, including Src family tyrosine kinases, Lck (resulting in impairment of Lck-mediated signalling events)  and Hck (resulting in its activation) . As the same signalling pathways are involved in FcγR-mediated phagocytosis by MDM, (reviewed in ), Nef may influence phagocytosis by binding to Hck or other relevant kinases or cytoskeletal proteins. To assess this hypothesis we investigated the direct effects of infection and replication of HIV-1 on macrophage function as well as the effect of Nef in the absence of other HIV-1 proteins. Phagocytosis of MAC was defective in MDM infected in vitro with either WT or nef-deleted strain of HIV-1. Experiments to specifically address both FcγR- and C′R-mediated phagocytosis (occurring via different signalling mechanisms ) by HIV-infected MDM showed similar levels of impairment. In our studies deletions within nef did not significantly alter the replicative capacity of HIV-1 in MDM (in agreement with previous reports [60,61], in contrast to other studies ). Therefore, our results suggest that decreased phagocytosis in vitro is predominantly a direct consequence of HIV-1 replication in MDM, and that Nef does not contribute to this impairment of MDM function. Unaltered levels and pattern of tyrosine phosphorylation of key proteins during FcγR-mediated phagocytosis by Nef-electroporated MDM confirmed that Nef on its own is not sufficient to inhibit the process of phagocytosis.
In conclusion, there is no impairment in phagocytosis by monocytes in whole blood from individuals infected with Δnef strains of HIV-1 in contrast to subjects infected with WT HIV-1, possibly reflecting an indirect effect of infection with HIV-1 resulting in altered actin polymerization in these cells. However, in vitro infection of MDM with nef-deleted strains and WT HIV-1 at relatively high multiplicity of infection both equally impair phagocytosis, indicating a direct effect of HIV-1 infection and replication on macrophage function, and suggesting that Nef does not directly impact on macrophage phagocytic activity.
The authors would like to thank Geza Paukovics for assistance with flow cytometric analysis, May Cleary for technical assistance with phalloidin assay, Mandy Dunne and Fiona Mitchell for performing viral load assays. We thank Dale McPhee for providing HIV-1C18 viral stock, Jenny Learmont and Mary Rose Birch for provision of SBBC samples.
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