Colony-Stimulating Factor 1 Receptor Antagonists Sensitize Human Immunodeficiency Virus Type 1-Infected Macrophages to TRAIL-Mediated Killing

ABSTRACT

Strategies aimed at eliminating persistent viral reservoirs from HIV-1-infected individuals have focused on CD4⁺ T-cell reservoirs. However, very little attention has been given to approaches that could promote elimination of tissue macrophage reservoirs. HIV-1 infection of macrophages induces phosphorylation of colony-stimulating factor 1 receptor (CSF-1R), which confers resistance to apoptotic pathways driven by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), thereby promoting viral persistence. In this study, we assessed whether CSF-1R antagonists (PLX647, PLX3397, and PLX5622) restored apoptotic sensitivity of HIV-1-infected macrophages in vitro. PLX647, PLX3397, and PLX5622 at clinically relevant concentrations blocked the activation of CSF-1R and reduced the viability of infected macrophages, as well as the extent of viral replication. Our data show that strategies targeting monocyte colony-stimulating factor (MCSF) signaling could be used to promote elimination of HIV-1-infected myeloid cells and to contribute to the elimination of persistent viral reservoirs.

IMPORTANCE As the HIV/AIDS research field explores approaches to eliminate HIV-1 in individuals on suppressive antiviral therapy, those approaches will need to eliminate both CD4⁺ T-cell and myeloid cell reservoirs. Most of the attention has focused on CD4⁺ T-cell reservoirs, and scant attention has been paid to myeloid cell reservoirs. The distinct nature of the infection in myeloid cells versus CD4⁺ T cells will likely dictate different approaches in order to achieve their elimination. For CD4⁺ T cells, most strategies focus on promoting virus reactivation to promote immune-mediated clearance and/or elimination by viral cytopathicity. Macrophages resist viral cytopathic effects and CD8⁺ T-cell killing. Therefore, we have explored clearance strategies that render macrophages sensitive to viral cytopathicity. This research helps inform the design of strategies to promote clearance of the macrophage reservoir in infected individuals on suppressive antiviral therapy.

INTRODUCTION

Macrophages are permissive to infection by primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) (1–3); have a prolonged life span (4, 5); and are widely distributed throughout the body in tissues like the lymph nodes (6), gut (7–9), central nervous system (10), and lung (11). Because macrophages are resistant to viral cytopathic effects, they have the potential to act as a viral reservoir in HIV-1-infected individuals on suppressive antiretroviral therapy (ART) (1, 12–17). Furthermore, infected macrophages harbor infectious HIV-1 particles in stable intracellular virus-containing compartments (VCCs) that are connected to the plasma membrane (18). These VCCs protect the archived virions from antibody-mediated neutralization (19) while enabling infection in trans at virological synapses with nearby T cells (13, 20). These synapses promote cell-to-cell transmission and a high multiplicity of infection that may impact antiviral efficacy (21). Despite a wealth of information regarding the viral and cellular factors that impact permissivity of macrophages to HIV-1 infection, it is unclear whether and to what extent tissue macrophages serve as a viral reservoir in individuals on suppressive ART. If macrophages can indeed serve as viral reservoirs, their elimination may require strategies distinct from those being used to purge CD4⁺ T-cell reservoirs.

Most of the attention on cellular reservoirs that support viral persistence, as well as strategies aimed at their elimination, has focused on CD4⁺ T cells and approaches to promote reactivation of HIV-1 from latency. Methodologies to induce reactivation of viral latency ultimately rely on the induction of viral cytopathicity and/or the elimination of the reactivated cell by host immunity so that the infected cell can be cleared. Similarly, approaches to eliminate the macrophage reservoir will need to overcome the inherent resistance of infected macrophages to viral cytopathicity. Recent studies have accordingly focused on identifying the underlying basis for cytopathic resistance and ways to circumvent this resistance (22).

HIV-1 infection of macrophages has been shown to affect their sensitivity to oxidative stress and to trigger apoptosis of bystander CD4⁺ and CD8⁺ T cells (23, 24). We previously demonstrated that HIV-1 infection of macrophages results in induction of the myeloid cell prosurvival cytokine monocyte colony-stimulating factor (MCSF) and the induction of MCSF-conferred resistance to apoptotic stimuli, thereby preserving the viability of the infected cell (25). The anticancer agent imatinib, which is a low-affinity inhibitor of the MCSF receptor, colony-stimulating factor 1 receptor (CSF-1R), was shown to inhibit MCSF signaling and to restore the sensitivity of HIV-1-infected macrophages to apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (25). We now extend this observation by examining the impact of higher-affinity CSF-1R antagonists on the sensitivity of HIV-1-infected macrophages to apoptosis. Our results indicate that inhibition of CSF-1R phosphorylation by CSF-1R antagonists restores the sensitivity of infected macrophages to apoptotic cell death by TRAIL, revealing a potential strategy to promote clearance of myeloid viral reservoirs in HIV-1-infected individuals.

MATERIALS AND METHODS

Reagents and antibodies.

PLX03, PLX647, PLX5622, and PLX3397 were provided as powder (Plexxikon Inc.) and subsequently solubilized in dimethyl sulfoxide (DMSO) (Sigma-Aldrich). CSF-1R antagonists and PLX03 were used at 10 μM with final DMSO concentrations of 0.1%. Soluble recombinant human TRAIL (rhTRAIL) (R&D) was used at 5 ng/ml. Imatinib mesylate (Santa Cruz Biotechnology) was used at 10 μM. Nevirapine was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, and used at supraphysiological concentration, 2 μM, to ensure that traditional viral replication was inhibited. Staurosporine (Sigma-Aldrich) was used at 1 μg/ml. MCSF was supplied by R&D Systems. Formaldehyde was obtained from Sigma-Aldrich. KC57-RD1 (Coulter Clone) and LIVE/DEAD fixable near-infrared (IR) dead-cell stain (Life Technologies) were used to detect HIV-1_gag⁺ and dead cells, respectively, by flow cytometry (LSR II; BD).

Cells.

Monocytes were obtained by leukapheresis from normal donors seronegative for HIV-1 and hepatitis B and were enriched by countercurrent centrifugal elutriation, as detailed previously (26). Highly purified untouched monocytes were further isolated by an indirect magnetic-labeling system, as instructed by the manufacturer (Miltenyi Biotec). Monocytes were differentiated into macrophages in complete medium comprised of Dulbecco's modified Eagle's medium (DMEM) (Gibco) containing 10% heat-inactivated human serum (Sera Care Life Sciences), 2 mM l-glutamine (Gibco), 10 μg/ml gentamicin (Sigma-Aldrich), and 6 ng/ml of human recombinant monocyte colony-stimulating factor (rhMCSF) (R&D Systems). Cells were seeded in 24-well plates (Corning) and cultured for 7 days at 37°C with 5% CO₂. The macrophages were then used for virus infections.

Viruses and infections.

Viral stocks were generated in 293T cells cotransfected with Lipofectamine 2000 (Invitrogen) and plasmids encoding the HIV-1 molecular clones and the vesicular stomatitis virus glycoprotein (VSV-G), using a 12:1 ratio of DNA. P121 HIV-1 ADA (HIV_ADA) was kindly provided by Mark Sharkey, and pNL43IeG-Nef⁺ (HIV_NL4-3–green fluorescent protein [GFP]) was obtained through the NIH AIDS Research and Reference Reagent Program. Virus-containing supernatants were harvested at 48 h and 72 h posttransfection and further purified over a 20% sucrose cushion, as previously described (27). Virus stocks were frozen in aliquots for single use after passing them through 0.45-μm filters and quantitated by measurement of reverse transcriptase (RT) activity and HIV-1 p24^gag by ELISA, according to the manufacturer's protocol (Beckman-Coulter). Macrophages were infected overnight (18 h) with 170 ng per well of either p24^gag of HIV_ADA or HIV_NL4-3-GFP (VSV-G pseudotyped). The input virus was washed off, and the macrophages were further cultured in 1.5 ml of complete medium lacking MCSF. VSV-G-pseudotyped viruses were used for infection of macrophages, as pseudotyping promotes far more efficient first-round infection of cells than nonpseudotyped HIV-1. As cells are infected synchronously, changes in host cell function are uniform and not obfuscated by a large background of uninfected cells.

Quantification of phospho-CSF-1R.

Complete medium was replaced by XVIVO10 (Lonza) to serum starve the macrophages overnight. Subsequently, the cells were pretreated for 2 h with CSF-1R antagonists and stimulated for 5 min with MCSF (100 ng/ml). Cell lysates were generated with RIPA buffer (Alfa Aesar) containing protease (Sigma-Aldrich) and phosphatase (Cell Signaling Technology) inhibitor cocktails and further concentrated through 50-kDa protein concentrators (Sartorius). The protein concentration was determined by DC protein assays (Bio-Rad), and lysates at 5 mg/ml were used to quantify the phosphorylated CSF-1R (phospho-CSF-1R) by the PathScan Phospho-MCSF-Receptor sandwich enzyme-linked immunosorbent assay (ELISA) (Cell Signaling Technology), as described by the manufacturer.

Analysis of viral infection by PCR.

Macrophages were washed in phosphate-buffered saline (PBS), incubated with DNAzol (Invitrogen) and dispersed by repeated pipetting, after which DNA was extracted as described previously (13). The total HIV-1 DNA copy number was determined with an Applied Biosystems 7700 real-time PCR system with C1r (5′-TCCCAGGCTCAGATCTGGTCTA-3′) and C4r (5′-CTTCCCTGATTGGCAGAACTAC-3′) primers and a 2n4n probe (5′-AGTGGCGAGCCCTCAGATGCTGC-3′). Copy number estimates of total HIV-1 DNA were determined using the ABI prism 7700 quantification software. The number of cell equivalents in DNA lysates was determined by PCR using primers directed to the CCR5 gene, as described previously (28).

Statistical analysis.

Statistical analyses were performed using GraphPad Prism, version 6.0 (GraphPad Software). One-way analysis of variance (ANOVA) was performed to determine whether the treatments with CSF-1R antagonists were having an impact on the cultures. Dunnett's multiple-comparison test was further implemented to statistically analyze the changes obtained from the treatment with PLX03, a compound that was not a CSF-1R antagonist.

RESULTS

Inhibition of MCSF-mediated phosphorylation of the CSF-1R by active CSF-1R antagonists.

We initially evaluated whether CSF-1R antagonists blocked the phosphorylation of the CSF-1R in primary human monocyte-derived macrophages (MDM). MDM were serum starved, preincubated with CSF-1R antagonists, and primed with MCSF, after which lysates were prepared for protein analysis (Fig. 1a). Upon ligand binding, CSF-1R dimerizes and undergoes tyrosine phosphorylation (29). Early responses in the activation of CSF-1R were measured by brief stimulation with MCSF, aiming to limit the late-stage internalization and lysosomal degradation of the receptor (30). As expected, phosphorylation of the CSF-1R was stimulated in response to MCSF treatment when MDM were preincubated with vehicle (DMSO) or with an inactive CSF-1R antagonist (PLX03) but was blocked when cells were pretreated with active CSF-1R antagonists (e.g., PLX647, PLX5622, PLX3397, or imatinib) (Fig. 1b). Comparable levels of phosphorylated CSF-1R were observed in mock- and PLX03-treated MDM, confirming that PLX03 is an inactive analog that does not impact the efficiency of CSF-1R phosphorylation in the presence of MCSF. All active CSF-1R analogs showed similar potencies in blocking the phosphorylation of the CSF-1R.

FIG 1.

PLX647, PLX3397, PLX5622, and imatinib inhibit the MCSF-dependent autophosphorylation of CSF-1R in MDM. (a) Schematic of the experimental design. The extent of CSF-1R phosphorylation was examined by ELISA with a CSF-1R- and a phosphotyrosine-specific antibody. (b) In each experiment, phosphorylated CSF-1R values obtained from the different treatments were normalized to the vehicle control (DMSO only) and graphed as percentages. Each dot corresponds to an independent experiment. Means and standard deviations are shown. The open circle identifies the treatment (PLX03) that was used in Dunnett's multiple-comparison test of one-way ANOVA to statistically analyze changes. ****, P < 0.0001.

CSF-1R antagonists reduce virus output and viability of infected macrophages.

We next examined the ability of CSF-1R antagonists to sensitize HIV-1-infected MDM to death by rhTRAIL. The compounds and rhTRAIL were added 1 day postinfection rather than prior to infection to maintain similar levels of infection in each system. MDM were cultured for 5 days, as illustrated in Fig. 2a, after which the macrophages were recovered and cellular DNA was extracted. To quantify the remaining macrophages following treatment with the different CSF-1R antagonists, the CCR5 copy number was assessed by PCR. The extent of virus production was determined from the level of viral reverse transcriptase activity in cell-free supernatants following incubation with the CSF-1R antagonists.

FIG 2.

CSF-1R antagonists PLX647, PLX3397, and PLX5622 reduce cell numbers, the extent of virus production, and the number of infected cells in HIV-1-infected macrophage cultures. (a) Experimental design. (b) Number of cell equivalents in DNA lysates after each treatment. (c) Virus output in cell-free supernatants quantified by RT activity (counts per minute) per microliter of supernatant. (d) Percentage of infected macrophages present in each culture following treatment with CSF-1R antagonists. The data are representative of the results of three independent experiments (means and standard deviations are shown). The open bars represent uninfected cultures, while the shaded bars represent infected cultures. The open circles identify the treatments (PLX03) that were used in Dunnett's multiple-comparison tests of one-way ANOVA to statistically analyze changes. ****, P < 0.0001; ***, P = 0.0001 to 0.001; **, P = 0.001 to 0.01; *, P = 0.01 to 0.05; ns, not significant. qPCR, quantitative PCR.

To ensure that the viral inoculum originally used to infect the MDMs was not confounding the viral readouts, infections were conducted in the presence of the nonnucleoside reverse transcriptase inhibitor nevirapine. Comparable levels of cell equivalents were detected in MDM that were not infected or that were infected in the presence of nevirapine (Fig. 2b). Even when infections were performed in the presence of vehicle, which allowed the spread of infection, the number of cells retrieved at the end of the culture was maintained. These results highlight the profound resistance of macrophages to HIV cytopathicity. Culture of infected macrophages with the inactive CSF-1R compound PLX03 did not affect macrophage viability. However, early treatment of HIV-1-infected macrophages with active CSF-1R antagonists led to a reduction in the number of myeloid cells in culture: PLX647 promoted a 75% decrease in cell numbers relative to PLX03-treated cultures, while PLX3397 decreased cell numbers by 55% and PLX5622 by 43%. These results indicate that active, but not inactive, CSF-1R antagonists sensitize HIV-1-infected macrophages to rhTRAIL-mediated apoptosis.

To establish whether active CSF-1R antagonists affected the extent of virus production from infected macrophages, cell-free supernatants were recovered after a 5-day incubation with the agents, and RT activity was measured. Since the antiretroviral control matched levels in uninfected cells (Fig. 2c), the detected RT activity quantified de novo-synthesized virions from productively infected macrophages as opposed to input virus (inoculum). Interestingly, HIV-1-infected macrophages treated with active CSF-1R antagonists 1 day postinfection showed a modest but consistent and statistically significant reduction in virus output compared to treatment with the inactive CSF-1R antagonist, PLX03. PLX647 promoted a 62% decrease in RT activity, while PLX3397 and PLX5622 caused a 30% and a 25% decrease, respectively. To distinguish whether the reduction in virus output was due to a reduction in the extent of virus output per cell or, alternatively, a reduction in the number of virus-producing cells, the proviruses were quantified under each condition. After normalization to cell equivalents, the percentage of macrophages harboring proviruses in PLX03-treated infected cultures was found to be comparable to that in cultures containing vehicle alone (Fig. 2d). However, treatment with an active CSF-1R antagonist (PLX647, PLX3397, or PLX5622) resulted in specific decreases in the proportion of macrophages containing HIV-1 proviruses by 88%, 64%, and 57%, respectively. These results indicate that active CSF-1R antagonists cause a reduction in virus output by promoting the death of infected macrophages through rhTRAIL.

CSF-1R antagonists reduce the frequency of productively infected macrophages.

To validate the above-described results at the single-cell level, we conducted single-cycle infections employing a VSV-G-pseudotyped, T-cell-tropic HIV-1 reporter virus. In these instances, the VSV-G protein promotes a first round of infection, but subsequent rounds do not occur, since the viruses produced harbor only T-cell-tropic HIV-1 envelopes incapable of infecting macrophages (31). Macrophages were infected with the reporter virus and subsequently treated with the CSF-1R antagonists and rhTRAIL (Fig. 3a). Five days later, the cells were fixed, and the percentage of GFP⁺ macrophages was analyzed by flow cytometry. The percentage of macrophages that had integrated viral DNA and that were producing viral particles was determined from the frequency of GFP-expressing cells. All active CSF-1R antagonists promoted a reduction in the percentage of GFP⁺ macrophages (50%, 47%, and 49% reductions in GFP⁺ macrophages by PLX647, PLX3397, and PLX5622, respectively) (Fig. 3b). The cytotoxic agent staurosporine, in comparison, caused a 60% reduction in GFP-expressing macrophages and a decrease in the number of acquired cells. Uninfected cells were used to account for intrinsic autofluorescence and for establishing the gates.

FIG 3.

CSF-1R antagonists PLX647, PLX3397, and PLX5622 reduce the percentage of productively infected macrophages. (a) Experimental design. (b) (Left) Frequency of GFP⁺ macrophages after treatment with the CSF-1R compounds. Staurosporine was used as a positive control. Each dot corresponds to an independent experiment. Infected and uninfected cultures are displayed in blue and black, respectively. Means and standard deviations are shown. The open circle identifies the treatment (PLX03) that was used in Dunnett's multiple-comparison test of one-way ANOVA to statistically analyze changes. (Right) Contour plots (2%) used to calculate the frequencies of singlet GFP⁺ macrophages for each condition. Each treatment contour plot (blue) was superimposed on the uninfected controls (black) to show the levels of autofluorescence. ****, P < 0.0001. FSC-H, forward scatter height.

CSF-1R antagonists decrease the levels of live HIV-1-infected macrophages.

Treatment of infected macrophages with CSF-1R antagonists decreased the number of HIV-1-infected macrophages by rhTRAIL. To evaluate whether CSF-1R antagonism had a greater impact on macrophages with higher levels of virus production (based on levels of RT activity in the supernatant and cell-associated HIV-1 gag), macrophages at the peak of infection were cultured with CSF-1R antagonists in the presence of rhTRAIL (Fig. 4a). At the end of the experiment, all the cells were stained with an amine-reactive dye to detect nonviable cells and with a fluorochrome-conjugated antibody to detect HIV-1 gag antigens. The cells were then gated on live gag⁺ cells. Relative to the inactive analog PLX03, PLX647 and PLX5622 promoted statistically significant reductions of 30% and 28% of such cells, respectively (Fig. 4b). The reduction in infected-cell viability caused by PLX3397 was evident but not significant.

FIG 4.

CSF-1R antagonists PLX647 and PLX5622 reduce the levels of live HIV-1_gag⁺ macrophages. (a) Experimental design. (b) Amine-reactive-negative macrophages expressing intracellular HIV-1_gag were detected by flow cytometry and are displayed as percentages of total singlets. Staurosporine was used as a positive control for killing. The bars represent the means of three independent experiments, and the error bars correspond to their standard deviations. The shaded bars represent infected cultures. The open circle identifies the treatment (PLX03) that was used in Dunnett's multiple-comparison test of one-way ANOVA to statistically analyze changes. ****, P < 0.0001; **, P = 0.001 to 0.01; *, P = 0.01 to 0.05; ns, not significant.

DISCUSSION

While ART has transformed the lives of individuals living with HIV-1, it does not cure infection. There is now a considerable research effort under way to identify strategies to eliminate the reservoirs in which the virus persists. As researchers embark on an effort to cure HIV-1 infection, a complete understanding of the viral reservoirs that support viral persistence is necessary. The nature of the cellular reservoirs is likely to dictate the strategies that will be employed against them. For the reservoir of latent proviruses in memory CD4⁺ T cells, “shock-and-kill” approaches aim to reactivate the latent virus with the hope that the cell will succumb to viral cytopathicity and/or to clearance by cell-mediated immunity (32). The latent memory CD4⁺ T-cell reservoir should be amenable to this approach, since infected CD4⁺ T cells are susceptible both to viral cytopathicity and to CD8⁺ T-cell killing (33). However, the resistance of macrophages to viral cytopathicity (13) and to CD8⁺ T-cell killing (34) may dictate alternative approaches to achieve elimination of viral reservoirs in macrophages.

In this study, we have explored whether HIV-1-infected macrophages might be eliminated by intervening in an important prosurvival pathway. We have previously demonstrated that HIV-1 activates the prosurvival cytokine MCSF to promote survival of infected macrophages in the face of cytopathic infection and apoptotic stimuli, such as TRAIL (25). That activation was observed to be dependent on the downregulation of the TRAIL receptor (TRAIL-R) and the upregulation of antiapoptotic genes, like the Bfl1 and Mcl1 genes. Other genes, like the Bcl-2 gene, may also play a role in that macrophage prosurvival pathway, as suggested for monocytes (35). Low-affinity antagonists of MCSF signaling (e.g., imatinib) increased the apoptotic sensitivity of HIV-1-infected macrophages (25). Here, we tested three novel antagonists of MCSF signaling (PLX647, PLX3397, and PLX5622) for the ability to restore the sensitivity of HIV-1-infected macrophages to apoptotic cell death by TRAIL. Compared to imatinib, these agents are more selective antagonists of MCSF signaling, specifically preventing phosphorylation and activation of the MCSF receptor, CSF-1R. Imatinib, in contrast, targets C-abl, c-kit, PDGFR, CSF-1R, and Lck (36). PLX647 targets CSF-1R and also c-kit, while PLX3397 blocks CSF-1R, c-kit, and possibly also PDGFRb (37). We assessed them in parallel with a compound (PLX03) that does not have inhibitory activity against the CSF-1R in either biochemical or cell-based assays but is similar to the CSF-1R antagonists in molecular weight and composition. PLX03 does not have appreciable activity against other kinases but has the same 7-azaindole scaffold that is the active component of numerous kinase inhibitors (38) and is present in the CSF-1R antagonists assessed in this study. As PLX03 does not exhibit antagonism to CSF-1R activation and does not impact HIV activity in macrophages, it demonstrates that the impact of these agents on HIV in macrophages was mediated by their effect on CSF-1R phosphorylation rather than via potential nonspecific, off-target effects of the agents on a process unrelated to CSF-1R phosphorylation.

As expected, treatment of HIV-1-infected macrophages with the inactive compound PLX03 did not affect the rates of phosphorylation of CSF-1R in macrophages following their stimulation with MCSF (Fig. 1). Treatment of HIV-1-infected macrophages with this inactive CSF-1R antagonist affected neither their viability (Fig. 2b) nor their ability to support HIV-1 replication (Fig. 1b and 2c and d). Therefore, no cellular toxicities were observed in vitro at the tested concentrations, as others have previously shown (38).

The CSF-1R antagonists PLX647, PLX3397, and PLX5622 impacted the viability of infected macrophages and their ability to support HIV-1 replication at 10 μM. Lower concentrations (1 μM and 3 μM) did not result in antiviral response in macrophages (data not shown). The inability of infected macrophages to phosphorylate the CSF-1R promoted their death upon treatment with TRAIL. In contrast, treatment of HIV-1-infected macrophages with the inactive compound PLX03 did not affect the rates of phosphorylation of CSF-1R in macrophages following their stimulation with MCSF (Fig. 1). Therefore, inhibition of macrophage viability and viral replication was a consequence of impaired CSF-1R activation and not the result of generalized impairment of host cell function by this class of agents.

Assessing the impact of CSF-1R antagonists on viral replication in HIV-1-infected macrophages poses several technical considerations. Differences in the extents of killing obtained in macrophages infected with distinct HIV-1 strains could have resulted from disparities in the number of rounds of viral replication and/or the time when the CSF-1R compounds were introduced into the culture (Fig. 3b and 4b). CSF-1R compounds sensitize infected macrophages to apoptosis but do not block new cycles of infection. Therefore, ongoing viral replication may have masked the results shown in Fig. 4b by introducing de novo infection into the culture and reducing the drugs' impact. The protective effect elicited by MCSF, which is induced by infection, may be more pronounced in cells at the peak of virus activity. MCSF induction, and as a consequence protection from TRAIL-mediated apoptosis, may therefore be less apparent in cells recently infected. We tried to control these variables by using VSV-G-pseudotyped virus to promote a more synchronous infection.

Inhibition of macrophage persistence may impact effects that extend beyond the reduction of the reservoir. Infected macrophages release inflammatory cytokines that can contribute to the aberrant inflammatory state that persists in the face of suppressive ART (39). MCSF has been shown to increase CD4 and CCR5 expression on uninfected macrophages (24), and this could provide additional substrates for virus replication. Conversely, it is not known whether inhibition of MCSF signaling might reduce coreceptor expression. While we did not examine whether these agents impacted coreceptor expression on macrophages, any potential effect on receptor expression would be unlikely to impact infection efficiency, since VSV-G-pseudotyped virus, which does not use CD4 or CCR5 for infection, was used for infection in our study. Depleting HIV-1-infected macrophages may result in the decline of cytokine and chemokine production and, hence, reduction of inflammation and immune cell recruitment, each of which has been associated with poor prognoses and ongoing viral replication (40). CSF-1R antagonists have been shown to reduce TNF-α in rodent models (38, 41). Reductions in interleukin 6 (IL-6) and inflammatory cell infiltration were also observed with PLX647 (38) and a decrease in IL-1β levels with PLX5622 and PLX3397 (41, 42). PLX5622 has also shown a decrease in inflammation in rheumatoid arthritis by impacting macrophage proliferation.

It remains to be determined whether inhibition of MCSF signaling is a viable approach for elimination of macrophage reservoirs in vivo. Inhibition of CSF-1R activation renders cells more susceptible to apoptotic ligands, such as TRAIL. CD4⁺ T cells and dendritic cells express TRAIL (43), but monocytes may be the most important source of TRAIL (44, 45). Plasma levels of TRAIL are increased in patients infected with HIV-1 (46) and are decreased, but not normalized, by antiretroviral therapy (45). Therefore, levels of TRAIL in aviremic individuals on effective ART may be suboptimal with regard to apoptotic clearance of macrophages if treated with CSF-1R antagonists. Our in vitro experiments attempted to create conditions under which TRAIL and CSF-1R antagonist concentrations were physiologic. Clinical trials have been performed in patients with recurrent glioblastoma by orally administering 1,000 mg of PLX3397 on a daily basis (47) and showed a median maximal concentration of 19.4 μM and a median value of 13 μM, values that were higher than our working concentration (10 μM). TRAIL has gained attention in oncology due to its specificity in killing tumor cells in rodents and monkeys while exhibiting minimal off-target toxicity (48, 49). In vitro treatment of primary lymphocytes from suppressed HIV-1-infected individuals with soluble TRAIL showed reductions in viral replication without drastic alterations in cytokine profiles (50). In our studies, we used concentrations of TRAIL that can be found in vivo in viremic HIV-1-infected individuals (46). Thus, the utility of CSF-1R antagonists in HIV-1 infection could be explored in the setting of clinical protocols for treatment of certain myeloproliferative conditions that employ CSF-1R antagonists. These small CSF-1R antagonists were designed to penetrate the blood-brain barrier and have been shown to reduce 95 to 99% of microglia in brain plaques in an Alzheimer′s disease mouse model treated with PLX5622 and PLX3397(41, 42), showing their ability to penetrate this important sanctuary site. Concomitant treatment with antiretrovirals, CSF-1R antagonists, and TRAIL could block viral replication and promote clearance of infected macrophages.