HIV-1 Tat Upregulates TREM1 Expression in Human Microglia

Key Points

• HIV infection increases microglial survival through TREM1 expression.

• HIV Tat increases the expression of TREM1 in human microglia.

• Tat-mediated TREM1 expression is dependent on TLR4 signaling and PGE₂.

Visual Abstract

Abstract

Because microglia are a reservoir for HIV and are resistant to the cytopathic effects of HIV infection, they are a roadblock for any HIV cure strategy. We have previously identified that triggering receptor expressed on myeloid cells 1 (TREM1) plays a key role in human macrophage resistance to HIV-mediated cytopathogenesis. In this article, we show that HIV-infected human microglia express increased levels of TREM1 and are resistant to HIV-induced apoptosis. Moreover, upon genetic inhibition of TREM1, HIV-infected microglia undergo cell death in the absence of increased viral or proinflammatory cytokine expression or the targeting of uninfected cells. We also show that the expression of TREM1 is mediated by HIV Tat through a TLR4, TICAM1, PG-endoperoxide synthase 2, PGE synthase, and PGE₂-dependent manner. These findings highlight the potential of TREM1 as a therapeutic target to eradicate HIV-infected microglia without inducing a proinflammatory response.

Introduction

Forty years after its discovery, HIV is still a major public health challenge. Antiretroviral therapy (ART) has transformed infection with HIV from a terminal illness to a chronic disease with improved life expectancy. However, ART is not a cure (1–4), and people living with HIV (PLWH) may still have a significant loss in virus-specific immune functions (5) and persistent cognitive impairment (6, 7).

Under suppressive ART, HIV remains detectable at low levels (8), and on ART interruption or failure, it can rapidly rebound to high pretherapy levels (9, 10). The source of this resurgent viremia is the viral reservoir that is seeded within days of primary infection (6, 7), and that is the main obstacle to an HIV cure. These HIV reservoirs persist in different anatomical compartments, including the CNS, where the efficacy of ART is reduced (8, 11, 12). Entry of HIV into the brain likely occurs via the trafficking of cell-free virus or infected monocytes, macrophages, and/or CD4⁺ T cells from peripheral blood across the blood-brain barrier (13–15). Within the CNS, brain parenchyma resident CD4⁺ T cells, perivascular macrophages, and microglia have been shown to be important reservoirs for HIV regardless of ART (16–22). Microglia are CNS-resident macrophages that mediate the innate and adaptive immune and repair responses to invading pathogens and injury, maintain neuronal circuits, and regulate energy homeostasis. Because they have a long life span (∼4 y), undergo cell division, and are resistant to HIV-mediated cytopathogenesis, they can function as a stable, long-term HIV reservoir (23) and are thus a potential source of resurgent viremia within the CNS of PLWH (24–27). CNS viremia is also associated with ART resistance, and HIV-associated neurocognitive disorders (HANDs) may result from the periodic emergence of HIV from latency (28–30).

Despite the importance of microglia as an HIV reservoir and in viral persistence, the mechanism(s) responsible for their resistance to HIV-mediated cytopathogenesis is not well understood. Although HIV does not encode for specific apoptosis inhibitor proteins, several HIV proteins, including Tat, upregulate the expression of antiapoptosis proteins, including BCL2, BCLXL, BIRC2 (cIAP-1), BIRC3 (cIAP-2), CFLAR (c-FLIP), CSF1 (MCP1), and XIAP (31–38), and directly modulate the mitochondrial pathway of apoptosis in myeloid cells (26, 38, 39). In addition, we and others have demonstrated that HIV infection of macrophages induces the expression of triggering receptor expressed on myeloid cells 1 (TREM1) (35–38), the silencing of which leads to BCL2L11 (BIM)-mediated disruption of the mitochondrial membrane potential and the induction of apoptosis (36, 38). Microglia also express TREM1, and its expression exerts an important role in the pathology of a number of neurological disorders, including, but not limited to, subarachnoid hemorrhage (40–43), Parkinson’s disease (44), and Alzheimer’s disease (45–47), as well as in bacterial CNS infections (48–50). In the latter, bacterial LPS activation of TLR4 signaling can induce the PGE₂-mediated upregulation of TREM1 expression (51), which then acts synergistically with TLRs and Nod-like receptors to amplify proinflammatory immune responses and promote cell survival (52–54).

Tat is an 86- to 101-residue protein expressed during the early stages of HIV replication that is secreted by HIV-infected cells and is detectable in the brains and cerebrospinal fluid of PLWH irrespective of ART status (55–57). Tat can interact with and signal through TLR4 on neighboring bystander cells (58–61), and it has been shown to have direct neurotoxic effects through the induction of neuronal apoptosis, the reduction of neuronal integrity, and the disruption of neuronal homeostasis (62–69), and indirect neurotoxic effects by acting as a viral chemokine to recruit monocytes/macrophages into active areas of CNS infection while also inducing them to release neurotoxic factors, including TNF, IL-6, IL-1β, and excess NO (60, 70–75). These proinflammatory cytokines, along with secreted Tat, can activate microglia, which can lead to the reactivation of latent HIV transcription (76). In this study, we analyzed the expression and role of TREM1 in response to HIV infection of microglia and delineated a potential role for Tat.

Materials and Methods

Microglia

Venous blood was drawn from HIV-seronegative healthy volunteers (aged between 18 and 65 y) at University of California San Diego Health Sciences using a protocol that was reviewed and approved by an Institutional Review Board (IRB) under the auspices of the Human Research Protections Program of the University of California San Diego (approval no. 180485AW) in accordance with the requirements of the Code of Federal Regulations on the Protection of Human Subjects (45 CFR 46 and 21 CFR 50 and 56) and were fully compliant with the principles expressed in the Declaration of Helsinki. All volunteers gave written informed consent before their participation, all samples were deidentified, and donors remained anonymous. Whole blood was also commercially obtained from Innovative Research (catalog no. [Cat#] IWB1NAE) under an IRB exemption from the University of South Dakota IRB (exemption no. IRB-22-209). Blood samples were assigned to experimental groups through simple random sampling. PBMCs were isolated from whole blood by density gradient centrifugation over Ficoll-Paque Plus (Cytiva). Human monocyte-derived microglia (MMGs) were differentiated from primary human monocytes as previously described (77–79). In brief, 6 × 10⁶ PBMCs/ml were incubated in MMG media (RPMI 1640 GlutaMAX supplemented with 100 µg/ml streptomycin; 100 U/ml penicillin [all Gibco]; 10 ng/ml each of CSF1, CSF2, and nerve growth factor β; and 100 ng/ml CCL2 and IL-34 [all PeproTech]) for 4 h, after which nonadherent cells were removed by aspiration. Adherent cells were then washed with Dulbecco’s PBS (Gibco) and further incubated in microglia media for 14 d at humidified 37°C, 5% CO₂ with media changes every 3 d before use.

HIV

HIV Ba-L was obtained through the National Institutes of Health AIDS Reagent Program from S. Gartner, M. Popovic, and R. Gallo (80, 81). Virus stocks were prepared as previously described (82). HIV infectivity was calculated as the 50% tissue culture infectious doses as described previously (83), and multiplicity of infection (MOI) was confirmed using TZM-bl cells from Dr. J.C. Kappes, Dr. X. Wu, and Tranzyme (84). MMGs were infected with HIV at 0.1 MOI for 4 h, washed, and then cultured for 28 d in MMG media before analysis.

Chemicals

Full-length HIV Tat (B.FR.83.HXB2; K03455.1) (85) was prepared on 0.5 mmol 4(hydroxymethyl)phenoxymethyl-copolystyrene-1% divinylbenzene-resin (Applied Biosystems) using an Applied Biosystems 433A Peptide Synthesizer with FastMoc Chemistry as previously described (86). Peptides were deprotected and cleaved from the support using trifluoroacetic acid supplemented with 10% (v/v) (methylsulfanyl)benzene, 5% (v/v) H₂O, and 5% (v/v) ethane-1,2-dithiol. Crude product was precipitated in 2-methoxy-2-methylpropane at −20°C, washed several times, filtered, and dried under vacuum. Tat was purified by reverse-phase HPLC as previously described (87). HPLC analysis was performed as previously described (86), amino acid analysis was performed on a Beckman Model 6300 Amino Acid Analyzer, mass spectrometry was carried out using an Ettan MALDI-TOF mass spectrometer (Amersham Biosciences), and concentration was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher) using a molar attenuation coefficient of 8480 M⁻¹⋅cm⁻¹ at 280 nm (88). Monoclonal anti-Tat Ab was purchased from Abcam (Cat# ab43014, RRID: AB_732970). LPS and EGTA were purchased from Sigma; trypsin was purchased from Gibco; and celecoxib, nimodipine, SC560, and tacrolimus were purchased from Selleck Chemicals.

Cell death and inflammatory markers

Apoptotic ssDNA was quantified using an ssDNA mAb (Cat# ALX-804-192, RRID: AB_10541559; Enzo Life Sciences) as described previously (69). Lactate dehydrogenase (LDH) activity of supernatants was measured using the LDH cytotoxicity detection kit (Takara Bio), and percent cytotoxicity was calculated according to the manufacturer’s protocol. PGE₂ was quantified in cell culture supernatants using a forward sequential competitive enzyme immunoassay kit (Cat# SKGE004B; R&D Systems). TNF and IL-10 were quantified using the Human TNF-α Quantikine ELISA Kit (Cat# STA00D; R&D Systems) and the Human IL-10 Quantikine ELISA Kit (Cat# S1000B; R&D Systems), respectively.

Small interfering RNA transfection

MMGs were transfected with Thermo Fisher Silencer Select small interfering RNA (siRNA) MYD88 (ID s14194), NFATC1 (ID s9470), RELA (ID s11915), TICAM1 (ID s45114), TLR4 (ID s14194), TREM1 (ID s28910), or negative control siRNA (siNS; Cat# 4390846) using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher) in Opti-MEM (Gibco) according to the manufacturer’s instructions. Forty-eight hours later, cells were analyzed for target gene silencing and used in experiments. Transfection efficiency was assessed using BLOCK-iT Alexa Fluor Red Fluorescent Control (Thermo Fisher) by flow cytometry.

Western blotting

Whole-cell lysates were prepared using 20 mM HEPES (Gibco), 150 mM NaCl (Fisher), 1 mM EDTA (Sigma) supplemented with 1% (v/v) Triton X-100 (Sigma), and 1% (v/v) Halt protease and phosphatase inhibitor mixture (Thermo Scientific). After 30 min on ice, cell homogenates were spun at 14,000 × g for 15 min, and supernatants were harvested. Cytoplasmic and nuclear fractions were prepared using the NE-PER nuclear and cytoplasmic extraction reagents according to the manufacturer’s protocol (Thermo Scientific). Lysates were resolved using 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol buffered polyacrylamide gels in MOPS buffer and transferred to 0.45-μm nitrocellulose membranes (Thermo Scientific), followed by detection with primary Abs, alkaline phosphatase–tagged secondary Abs (Invitrogen; Cat# WP20007, RRID: AB_2924319, or Cat# WP20006, RRID: AB_2924320), and 0.25 mM CDP-Star supplemented with 5% (v/v) Nitro-Block II (both from Applied Biosystems). The following primary Abs were used: anti-TREM1 (Cat# ab90808, RRID: AB_2050414; Abcam), anti-BAD (Cat# 9268S, RRID: AB_823433), anti-BAX (Cat# 5023, RRID: AB_2744530), anti-BCL2 (Cat# 15071, RRID AB_2744528), anti-BCLXL (Cat# 2764, RRID: AB_2228008), anti-H3C1 (Cat# 4499, RRID: AB_10544537), anti-MYD88 (Cat# 4283, RRID: AB_10547882), anti-NFATC1 (Cat# 8032, RRID: AB_10829466), anti–PG-endoperoxide synthase 1 (Cat# 9896, RRID: AB_10860249), anti-PTGS2 (Cat# 12282, RRID: AB_2571729), anti-RELA (Cat# 8242, RRID: AB_10859369), anti-TICAM1 (Cat# 4596, RRID: AB_2256555), and anti-TLR4 (Cat# 38519, RRID: AB_2924306) from Cell Signaling Technology; anti–phospho-NFATC1 Ser¹⁷² (Cat# MAB5640, RRID: AB_10719429), anti-PGE synthase (PTGES) (Cat# NBP3-15084, RRID: AB_2925176), and anti-PTGES3 (Cat# NB110-96879, RRID: AB_1260817) from Novus; and anti-ACTB (Cat# A2228, RRID: AB_476697) from Sigma. Relative densities of the target bands were compared with ACTB (for whole-cell lysates and cytoplasmic fractions) or H3C1 (for nuclear fractions) and were calculated using Fiji (RRID: SCR_002285). Each data point was normalized to the vehicle and then log₂ transformed.

Quantitative PCR

Total viral RNA was extracted directly from cell culture supernatants using the QIAamp viral RNA Mini kit (Qiagen), and measurement of HIV transcripts was performed as previously described (82, 89). Total RNA was isolated from cell pellets using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. RNA concentration was assessed using a NanoDrop 1000. cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using a Roche LightCycler 480 with the TaqMan Fast Advanced Master Mix and commercial probes for the targets IL1B (Hs01555410_m1; FAM-MGB), IL6 (Hs00174131_m1; FAM-MGB), PTGES (Hs00610420_m1; FAM-MGB), PTGES3 (Hs04187819_g1; FAM-MGB), PTGS1 (Hs00377726_m1; FAM-MGB), PTGS2 (Hs00153133_m1; FAM-MGB), TNF (Hs00174128_m1; FAM-MGB), TREM1 (Hs00218624_m1; FAM-MGB), and the reference gene POLR2A (Hs00172187_m1; VIC-MGB) (all Applied Biosystems). Relative quantification of the target gene expression compared with reference gene expression was performed using the Pfaffl method (90). Data were then normalized to the vehicle and log₂ transformed.

Statistics

Sample size (n) was determined using a two-sample two-sided equality test with power (1 − β) = 0.8, α = 0.05, and preliminary data where the minimum difference in outcome was at least 70%. Data were assessed for symmetry, or skewness, using Pearson’s skewness coefficient. Normalized ratiometric data were log₂ transformed. Comparisons between groups were performed using the paired, two-tailed Student t test. In all experiments, differences were considered significant when p was <0.05.

Results

HIV infection of microglia prolongs survival

Having previously identified that HIV infection promotes macrophage survival (38), we designed our initial experiments to determine whether HIV infection of microglia results in increased survival. We infected MMGs with HIV for 28 d, during which time we assessed HIV replication kinetics and apoptosis. Uninfected MMGs showed low levels of apoptosis with a maximum observed mean (± SD) of 16.2 ± 2.3% of cells displaying formamide-sensitive ssDNA (a specific indicator of apoptosis) (91) by day 28 (Fig. 1A). HIV infection of MMGs was associated with a 74.7% decrease in this value with a mean of 4.1 ± 1.0% exhibiting formamide-sensitive ssDNA (p = 0.003; Fig. 1A). HIV-infection of MMGs also resulted in a 78.1% reduction in cytotoxicity (17.4 ± 3.6% versus 4.2 ± 1.6%; p = 0.013; Fig. 1B) by day 28 d postinfection as assessed using LDH release. This observed increase in cell survival of HIV-infected MMGs (HIV-MMGs) corresponded with a decrease or little change in the expression of the proapoptotic proteins BAD and BAX and an increase in the expression of the antiapoptotic BCLXL, BCL2, and TREM1 (Fig. 1C). To elucidate a role for TREM1 in the survival of HIV-MMGs, we silenced TREM1 using RNA interference (RNAi; Fig. 1D). TREM1 silencing had no effect on uninfected MMG survival (Fig. 1E). In contrast, compared with the siNS-transfected HIV-MMGs, TREM1 silencing in HIV-MMGs significantly increased the presence of formamide-sensitive ssDNA (4.4 ± 1.8% versus 85.6 ± 5.8%; p = 0.00003) and LDH release (5.5 ± 2.4% versus 85.5 ± 5.9% cell death; p = 0.00003) (Fig. 1E). Importantly, TREM1 silencing did not increase viral transcription, nor did it induce an increase in proinflammatory cytokine mRNA expression (Fig. 1E).

FIGURE 1.

HIV infection increases microglial survival. MMGs were left uninfected or infected with 0.1 MOI HIV for 28 d. n = 4. (A) Cells were fixed and permeabilized, and the percentage of cells with apoptotic ssDNA was quantified by ELISA. Individual points indicate mean values from three technical replicates performed from each of four biological replicates. Lines follow grand mean values (± SD) of the four biological replicates. (B) Aliquots of supernatants were spectrophotometrically assayed for LDH activity. Individual points indicate mean values from three technical replicates performed from each of four biological replicates. Lines follow grand mean values (± SD) of the four biological replicates. (C) At day 28, cells were harvested. Left, Western blots of BCLXL, BCL2, TREM1, BAD, and BAX using Abs against BCLXL, BCL2, TREM1, BAD, BAX, and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graph represents the relative intensity of each band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. (D) At day 28, cells were transfected with TREM1 siRNA (siTREM1) or siNS. n = 4. Left, Representative Western blots of TREM1 using Abs against TREM1 and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graph represents the relative intensity of each band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. (E) Cells and supernatants from (D) were harvested and assayed for apoptotic ssDNA, LDH, and HIV gag RNA release and TNF and IL-1β mRNA expression. Each symbol represents the mean of an individual biological replicate performed in triplicate; small horizontal lines indicate the grand means of four biological replicates ± SD. Statistical analyses were performed using two-tailed paired t tests, *p < 0.05.

Next, we determined whether HIV Tat influenced the expression of proapoptotic and antiapoptotic proteins in uninfected MMGs. Tat dose-dependently increased the expression of BCL2 and BCLXL (Fig. 2A, 2B) while simultaneously decreasing the expression of both BAX and BAD (Fig. 2C, 2D). Tat also dose-dependently increased the expression of TREM1 (Fig. 2E). To rule out the role of any potential contaminant(s) in the synthetic Tat protein preparations, we analyzed the ability of the Tat preparations to induce the release of proinflammatory TNF and immunosuppressive IL-10 from MMGs after the preparations were incubated with either trypsin or a neutralizing anti-Tat Ab. Both treatments abolished the release of TNF and IL-10 in response to Tat while having no effect on the LPS-mediated expression (Fig. 2F). Moreover, the anti-Tat Ab completely prevented the Tat-mediated increase in TREM1 expression (Fig. 2G). These data indicate that the responses observed were specific to Tat and not caused by endotoxin contamination.

FIGURE 2.

HIV Tat increases the microglial expression of BCL2, BCLXL, and TREM1. (A–E) MMGs were exposed to HIV Tat. After 24 h, cells were harvested and assayed for BCL2 (A), BCLXL (B), BAX (C), BAD (D), and TREM1 (E). In (A)–(E), (left) a representative Western blot of respective targets using Abs against target and ACTB (the internal reference) for a single biological replicate; (right) densitometric analysis of blots. Graph represents the relative intensity of each band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. Statistical analyses were performed using two-tailed paired t tests, *p < 0.05. (F) A total of 10 ng/ml Tat or 100 ng/ml LPS was incubated with trypsin, an anti-Tat IgG, or an isotype IgG (Iso IgG) for 1 h at 37°C before being added to MMGs. After 24 h, TNF and IL-10 release was quantified by ELISA. Each symbol represents the mean of an individual biological replicate performed in triplicate; small horizontal lines indicate the grand means of four biological replicates ± SD. Statistical analyses were performed using two-tailed paired t tests, *p < 0.05. The dashed lines represent the limit of detection in each assay. (G) Tat was incubated with an anti-Tat IgG or an isotype IgG for 1 h at 37°C before being added to MMGs. After 24 h, cells were harvested. Top, Representative Western blots of TREM1 using Abs against TREM1 and ACTB (the internal reference) from one biological replicate. Bottom, Densitometric analysis of blots. Graph represents the relative intensity of each band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. Statistical analyses were performed using two-tailed paired t tests, *p < 0.05.

TREM1 is required for microglia to survive Tat exposure

To address the role of TREM1 in MMG survival after Tat exposure, we again silenced TREM1 using siRNA (Fig. 3A) with the hypothesis that if TREM1 is involved in promoting cell survival, then exposing TREM1-silenced cells to Tat should result in their death. Importantly, exposing siNS-transfected cells to Tat did not result in increased cell death (p > 0.28; Fig. 3B), nor did TREM1 silencing of uninfected MMGs (p > 0.13; Fig. 3B). Conversely, and similar to HIV-infected macrophages (38), TREM1-silenced MMGs exposed to Tat exhibited a high percentage of cells possessing apoptotic ssDNA (mean 68.0 ± 10.4%; p = 0.0009; Fig. 3B) and an overall increase in cell death (mean 70.4 ± 9.0%; p = 0.0003). TREM1 silencing also suppressed the Tat-induced upregulation of BCL2 and BCLXL expression (Fig. 3C, 3D) and inhibited the Tat-mediated downregulation of BAD and BAX expression (Fig. 3E, 3F).

FIGURE 3.

HIV Tat-mediated increase in BCL2 and BCLXL expression is dependent upon TREM1. MMGs were transfected with TREM1 siRNA (siTREM1) or siNS for 48 h, after which they were treated with Tat for a further 24 h. n = 4. (A) Left, Representative Western blots of TREM1 using Abs against TREM1 and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graph represents the relative intensity of each band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. (B) Left, Cells were fixed and permeabilized, and the percentage of cells with apoptotic ssDNA was quantified by ELISA. Right, Aliquots of supernatants were spectrophotometrically assayed for LDH activity. Each symbol represents the mean of an individual biological replicate performed in triplicate; small horizontal lines indicate the grand means of four biological replicates ± SD. (C–F) Left, Representative Western blots of BCL2 (C), BCLXL (D), BAD (E), or BAX (F) using Abs against BCL2, BCLXL, BAD, BAX, and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graphs represent the relative intensity of each target band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. Statistical analyses were performed using two-tailed paired t tests, *p < 0.05.

Tat upregulates TREM1 through TLR4 activation

Because Tat induces the expression of TNF and IL-10 from human monocytes through engagement of TLR4 and subsequent signaling through MYD88 and TICAM1 (TRIF) (58), and LPS upregulates TREM1 expression in macrophages through TLR4 signaling (92), we sought to delineate the role of TLR4 in the Tat-mediated upregulation of TREM1 in MMGs using TLR4 silencing (Fig. 4A). TLR4 silencing abolished the Tat-induced increase in TREM1 expression (Fig. 4B). Activation of TLR4 elicits two signaling cascades; the first involves the TIRAP and MYD88 adaptor proteins and is induced at the plasma membrane, whereas the second involves TRAM and TICAM1 and begins in early endosomes after TLR4 endocytosis. Because Tat binding to TLR4 leads to the activation of both of these signaling pathways (58), we next investigated which pathway was responsible for the Tat-mediated upregulation of TREM1 using MYD88 and TICAM1 silencing (Fig. 4C). TICAM1 silencing, but not MYD88 silencing, resulted in significant attenuation of TREM1 expression in response to Tat (Fig. 4C). Significantly, silencing of TLR4 inhibited both IL-10 and TNF responses to Tat, whereas both TICAM1 and MYD88 silencing inhibited the IL-10 response (Fig. 4D). However, MYD88, but not TICAM1, silencing inhibited the TNF response, consistent with the role of the myddosome in transmitting proinflammatory signals (93).

FIGURE 4.

HIV Tat-mediated TREM1 expression in microglia is dependent upon TLR4. (A) MMGs were transfected with TLR4 siRNA (siTLR4) or siNS for 48 h. Left, Representative Western blots of TLR4 using Abs against TLR4 and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graphs represent the relative intensity of each target band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. (B) Cells from (A) were treated with Tat for 24 h. Left, Representative Western blots of TREM1 using Abs against TREM1 and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graphs represent the relative intensity of each target band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. (C) MMGs were transfected with siRNA against MYD88 (siMYD88), TICAM1 (siTICAM1), or siNS for 48 h, after which they were treated with Tat for a further 24 h. Left, Representative Western blots of MYD88, TICAM1, and TREM1 using Abs against MYD88, TICAM1, TREM1 and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graphs represent the relative intensity of each target band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. (D) Supernatants from cells in (B) and (C) were assayed for TNF and IL-10 by ELISA. The dashed lines represent the limit of detection in each assay. Each symbol represents the mean of an individual biological replicate performed in triplicate; small horizontal lines indicate the grand means of four biological replicates ± SD. Statistical analyses were performed using two-tailed paired t tests, *p < 0.05.

Tat-mediated TREM1 expression is dependent on PGE₂

The LPS-induced upregulation of TREM1 expression in macrophages is dependent on the induction of PGE₂ metabolism from arachidonic acid by PTGS1 (also known as cyclooxygenase [COX] 1) and PTGS2 (also known as COX-2). Both PTGS isozymes catalyze the oxygenation of arachidonate to PGG₂ and reduce PGG₂ to PGH₂, which is then metabolized to PGE₂ by one of three PTGESs: the constitutively expressed PTGES3 (also known as cPGES and primarily coupled with the constitutively expressed PTGS1), the inducible PTGES (also known as mPGES-1 and primarily coupled with the inducible PTGS2), and PTGES2 (also known as mPGES-2 and which has no PTGS isoform specificity). Because Tat induces PTGS2 expression and activity in astrocytes (94), we evaluated whether Tat induces the upregulation of PTGS1 and/or PTGS2 in MMGs. Tat induced a dose-dependent increase in both PTGS1 and PTGS2 mRNA expression (Fig. 5A) that mirrored an increase in their protein expression (Fig. 5B). We also looked at the expression of PTGES and PTGES3 in response to Tat and found that Tat induced an increase in PTGES mRNA and protein expression while having a minimal effect on the expression of PTGES3 mRNA and protein levels (Fig. 5C, 5D). The observed increase in PTGS2 and PTGES expression also correlated with a dose-dependent increase in PGE₂ release from MMGs (Fig. 5E), which was completely inhibited by anti-Tat Abs (Fig. 5F). To confirm that the Tat-mediated increase in PTGS1, PTGS2, PTGES3, and PTGES expression and the synthesis of PGE₂ were mediated via TLR4 signaling, we again used TLR4 silencing. TLR4 silencing abolished the Tat-induced increase in PTGS1, PTGS2, PTGES3, and PTGES protein expression (Fig. 5G), as well as the Tat-mediated increase in PGE₂ release from MMGs (Fig. 5H).

FIGURE 5.

HIV Tat increases the expression of PTGS2, PTGES, and PGE₂ in microglia. (A–E) MMGs were treated with Tat for various time points. At 6 h, cells were harvested and analyzed for PTGS1, PTGS2 (A), PTGES, and PTGES3 (C) mRNA by quantitative PCR. Each symbol represents the mean of an individual biological replicate performed in triplicate; small horizontal lines indicate the grand means of four biological replicates ± SD. At 24 h, cells were harvested, lysed, and analyzed by Western blot for PTGS1, PTGS2 (B), PTGES, and PTGES3 (D) expression. Left, Representative Western blots of PTGS1, PTGS2, PTGES, and PTGES3 using Abs against PTGS1, PTGS2, PTGES, PTGES3, and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graphs represent the relative intensity of each target band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. At 24 h, supernatants were harvested and PGE₂ was quantified by competitive enzyme immunoassay (E). Each symbol represents the mean of an individual biological replicate performed in triplicate; small horizontal lines indicate the grand means of four biological replicates ± SD. (F) Tat was incubated with an anti-Tat IgG or an isotype control IgG (Iso IgG) for 1 h at 37°C before being added to MMGs. After 24 h, PGE₂ was quantified by competitive enzyme immunoassay. Each symbol represents the mean of an individual biological replicate performed in triplicate; small horizontal lines indicate the grand means of four biological replicates ± SD. (G) MMGs were transfected with TLR4 siRNA (siTLR4) or siNS for 48 h and then treated with Tat for a further 24 h, after which cells and supernatants were harvested. Left, Representative Western blots of TLR4, PTGS1, PTGS2, PTGES, and PTGES3 using Abs against TLR4, PTGS1, PTGS2, PTGES, PTGES3 and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graphs represent the relative intensity of each target band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. (H) PGE₂ was quantified in supernatants from (G) by competitive enzyme immunoassay. Each symbol represents the mean of an individual biological replicate performed in triplicate; small horizontal lines indicate the grand means of four biological replicates ± SD. (I–K) MMGs were pretreated with SC560 or celecoxib for 1 h before exposure to 10 ng/ml Tat. After 24 h, supernatants were harvested and PGE₂ was quantified by competitive enzyme immunoassay (I). Each symbol represents the mean of an individual biological replicate performed in triplicate; small horizontal lines indicate the grand means of four biological replicates ± SD. Cells were also harvested and TREM1 mRNA was quantified using quantitative PCR (J). Each symbol represents the mean of an individual biological replicate performed in triplicate; small horizontal lines indicate the grand means of four biological replicates ± SD. TREM1 and TLR4 protein expression were assessed by Western blot (K). Left, Representative Western blots of TLR4 and TREM1 using Abs against TLR4, TREM1, and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graphs represent the relative intensity of each target band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. Statistical analyses were performed using two-tailed paired t tests, *p < 0.05.

We next sought to determine whether the Tat-induced upregulation of TREM1 expression is mediated through the induction of PGE₂ by PTGS1 or PTGS2 using inhibitors for PTGS1 (SC560) and PTGS2 (celecoxib). Celecoxib significantly repressed PGE₂ synthesis (Fig. 5I) and TREM1 expression (Fig. 5J, 5K) in response to Tat, whereas SC560 did not. Importantly, neither celecoxib nor SC560 had any effect on TLR4 expression (Fig. 5K). Collectively, these data suggest that the Tat-mediated increase in TREM1 expression is dependent on PGE₂ synthesis mediated through the PTGS2 and PTGES axis.

Tat-mediated TREM1 expression is dependent on Ca²⁺ flux and NFATC1

Because we and others have previously shown that NF-κB transcriptionally regulates the expression of TREM1 in macrophages in response to LPS or Tat (36, 38, 95), we used RNAi for the NF-κB subunit RELA (Fig. 6A) to investigate whether the Tat-mediated TREM1 expression in MMGs is dependent on NF-κB. As expected, RELA silencing resulted in the inhibition of TREM1 expression in response to Tat exposure (Fig. 6B). However, RELA silencing failed to have an impact on Tat-mediated PTGS1 or PTGS2 expression (Fig. 6C) and failed to inhibit Tat-mediated PGE₂ production (Fig. 6D). These data suggest that although the expression of TREM1 is RELA dependent, and by extension NF-κB dependent, the Tat-mediated expression of PTGS1 and PTGS2 and subsequent PGE₂ synthesis that is required for the expression of TREM1 in MMGs is not. Because extracellular Tat induces the expression of PTGS2 in astrocytes through a mechanism involving NFAT and AP-1 transcription factors (94), we looked at the role of NFAT in the Tat-mediated expression of PTGS1 and PTGS2 and subsequent PGE₂ synthesis. NFAT proteins are Ca²⁺-regulated transcription factors that are heavily phosphorylated through the synergistic actions of casein kinase 1, glycogen synthase kinase 3, and dual-specificity tyrosine phosphorylation–regulated kinase (96–98). This phosphorylation masks their nuclear localization sequence and exposes their nuclear export sequence, resulting in the cytoplasmic localization of NFATs (96, 99). When the intracellular Ca²⁺ concentration increases, the calcium- and calmodulin-dependent serine/threonine protein phosphatase calcineurin is activated. This dephosphorylates NFAT, resulting in the exposure of the nuclear localization sequence, the masking of the nuclear export sequence, and the subsequent nuclear translocation of NFAT and the transcription of NFAT target genes (96, 99). Because Tat induces a rapid and transient Ca²⁺ influx in monocytes, macrophages, and microglia (74, 100, 101), we analyzed the effect of Tat on NFATC1 phosphorylation and localization. Tat exposure of MMGs resulted in the dephosphorylation (Fig. 7A) and nuclear accumulation (Fig. 7B) of NFATC1. We then investigated whether NFATC1 played a role in the Tat-mediated expression PTGS1 and PTGS2 and subsequent PGE₂ synthesis using RNAi for NFATC1 (Fig. 7C). NFATC1 silencing completely inhibited Tat-mediated PTGS1 and PTGS2 expression, as well as Tat-mediated PGE₂ synthesis.

FIGURE 6.

HIV Tat-mediated PGE₂ expression in microglia is not dependent on NF-κB. MMGs were transfected with RELA siRNA (siRELA) or siNS for 48 h, after which they were treated with Tat or vehicle for a further 24 h (n = 4). (A) Left, Representative Western blots of RELA using Abs against RELA and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graphs represent the relative intensity of each target band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. (B) Left, Representative Western blots of TREM1 using Abs against TREM1 and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graphs represent the relative intensity of each target band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. (C) Left, Representative Western blots of PTGS1 and PTGS2 using Abs against PTGS1, PTGS2, and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graphs represent the relative intensity of each target band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. (D) Supernatants were harvested, and PGE₂ was quantified by competitive enzyme immunoassay. Each symbol represents the mean of an individual biological replicate performed in triplicate; small horizontal lines indicate the grand means of four biological replicates ± SD. Statistical analyses were performed using two-tailed paired t tests, *p < 0.05.

FIGURE 7.

HIV Tat-mediated PGE₂ expression in microglia is dependent on NFATC1. (A and B) MMGs were treated with Tat or vehicle for 6 h. (A) Cells were harvested and analyzed for Ser¹⁷² phosphorylated NFATC1 by Western blot. A representative Western blot from one biological replicate using Abs against Ser¹⁷² phosphorylated NFATC1, total NFATC1, and ACTB is shown (n = 4). (B) Cells were harvested, and cytoplasmic and nuclear fractions were subjected to Western blot analysis using Abs against NFATC1, ACTB, and H3. Representative blots from one biological replicate are shown (n = 4). (C–E) MMGs were transfected with NFATC1 siRNA (siNFATC1) or siNS for 48 h, after which they were treated with Tat or vehicle for a further 24 h. (C) Left, Representative Western blot of NFATC1 using Abs against NFATC1 and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graphs represent the relative intensity of each target band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. (D) Left, Representative Western blots of PTGS1 and PTGS2 using Abs against PTGS1, PTGS2, and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graphs represent the relative intensity of each target band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. (E) Supernatants were harvested and PGE₂ was quantified by competitive enzyme immunoassay. Each symbol represents the mean of an individual biological replicate performed in triplicate; small horizontal lines indicate the grand means of four biological replicates ± SD. Statistical analyses were performed using two-tailed paired t tests, *p < 0.05.

Because NFAT proteins are Ca²⁺-regulated transcription factors that depend on calcineurin to be dephosphorylated, we then investigated the role of calcium and calcineurin in the Tat-mediated expression of PTGS1 and PTGS2. We and others have previously demonstrated that Tat induces a transient Ca²⁺ flux in human monocytes through l-type Ca²⁺ channels, and that this Ca²⁺ flux is totally blocked when cells are stimulated in the presence of nimodipine (74, 102). Therefore, we pretreated MMGs with nimodipine before exposing MMGs to extracellular Tat. Nimodipine inhibited the Tat-triggered expression of PTGS1 and PTGS2 (Fig. 8A, 8B), the synthesis of PGE₂ (Fig. 8C), and the expression of TREM1 (Fig. 8A, 8B), indicating that the expression of TREM1 is Ca²⁺ dependent. To further confirm the role of extracellular Ca²⁺ in the Tat-mediated expression of PTGS1 and PTGS2 and the synthesis of PGE₂, we used the Ca²⁺ chelator EGTA to remove Ca²⁺ from the medium and then analyzed the response of MMGs to Tat. EGTA inhibited the expected Tat-triggered response (Fig. 8A–C). Similarly, the calcineurin inhibitor tacrolimus also inhibited the Tat-mediated expression of PTGS1, PTGS2, and TREM1 (Fig. 8A–C). Importantly, inhibition of either Ca²⁺ flux or calcineurin inhibited the Tat-mediated TNF response while having no effect on the IL-10 response (Fig. 8D), in agreement with previous data (102–104).

FIGURE 8.

HIV Tat-mediated TREM1 expression in microglia is dependent on Ca²⁺-dependent PGE₂ production. MMGs were pretreated with 10 ng/ml tacrolimus, 1 µM nimodipine, or 2 mM EGTA for 1 h, after which they were treated with Tat. (A) After 6 h, cells were harvested and analyzed for PTGS1, PTGS2, and TREM1 mRNA by quantitative PCR. Each symbol represents the mean of an individual biological replicate performed in triplicate; small horizontal lines indicate the grand means of four biological replicates ± SD. (B) After 24 h, cells and supernatants were harvested. Lysates were subjected to Western blot analysis of PTGS1, PTGS2, and TREM1. Left, Representative Western blots of PTGS1, PTGS2, and TREM1 using Abs against PTGS1, PTGS2, TREM1, and ACTB (the internal reference) from one biological replicate. Right, Densitometric analysis of blots. Graphs represent the relative intensity of each target band to the corresponding ACTB signal. Each symbol represents an individual biological replicate; small horizontal lines indicate the means of four biological replicates ± SD. (C) Supernatants were assessed for PGE₂ content by competitive enzyme immunoassay. Each symbol represents the mean of an individual biological replicate performed in triplicate; small horizontal lines indicate the grand means of four biological replicates ± SD. (D) TNF and >IL-10 release was assayed by ELISA. Each symbol represents the mean of an individual biological replicate performed in triplicate; small horizontal lines indicate the grand means of four biological replicates ± SD. Statistical analyses were performed using two-tailed paired t tests, *p < 0.05. The dashed line represents the limit of detection in each assay.

Discussion

Despite the success of ART, it does not eliminate HIV, and there thus remains an unmet demand for the development of novel approaches that will lead to viral eradication. Current attention in HIV cure research focuses on reactivation of HIV from CD4⁺ T cells (105). Few studies address the myeloid cell reservoirs, including those found in the CNS, despite the need to clear these being crucial in any cure strategy. Moreover, because the immune and inflammatory responses mediated by HIV-infected microglia and perivascular macrophages contribute to neuronal dysfunction and drive the pathogenesis of HAND (106, 107), any strategy that reactivates HIV and/or leads to an increase in inflammatory cytokines and neurotoxin release in this environment is undesirable (23, 108). In addition, the very different nature of HIV infection of these cells, combined with their resistance to both HIV-mediated cytopathogenesis and CD8⁺ T cell killing, means that novel approaches are required to eliminate virus from macrophages and microglia while not exacerbating neuronal pathology (23).

The present data demonstrate that HIV infection of microglia increases cell survival and upregulates TREM1 expression, and that TREM1 silencing results in death of the infected, but not the uninfected, cells in the absence of proinflammatory cytokine expression or increased viral transcription. Moreover, we show that Tat upregulates TREM1 expression in MMGs via TLR4 and TICAM1 signaling through a mechanism that is inhibited by the PTGS2 inhibitor celecoxib. Supporting these findings, previous reports have demonstrated that Tat can signal through TLR4 (109) and induce PTGS2 expression and PGE₂ synthesis in astrocytoma cell lines (94), that TLR4 signaling can upregulate PTGS2 and PTGES expression in microglia (110), and that the induction of TREM1 expression is dependent on inducible PTGS2 and the synthesis of PGE₂ (51, 111, 112). In addition, PTGS2 overexpression during inflammatory reactions increases cellular resistance to apoptosis (113, 114), and in this article, we show that Tat-induced PTGS2 expression and activity correlate with MMG resistance to Tat-induced apoptosis. These data are also consistent with previous studies that demonstrate that although HIV infection is cytopathogenic for most CD4⁺ T cells, HIV infection of human microglia is not (78); however, HIV infection does result in an increase in secreted glutamate and other proinflammatory factors that can contribute to the death of bystander neurons and the recruitment of immune cells (115). Moreover, the survival of HIV-infected microglia permits the establishment of a latent reservoir that may exist for months to years (105, 116). TREM1 is thus a potential target to enhance clearance of HIV-infected microglia while minimizing inflammation and HIV transcription.

The chronic neuroinflammation that can occur during HIV infection can lead to the abnormal activation of microglia, a hallmark not only of HAND but also of a diverse set of neurological diseases (117, 118). This activation of microglia can induce endogenous TREM1 expression (119), and in models of ischemic stroke (120, 121), subarachnoid hemorrhage (41, 43, 122), intracerebral hemorrhage (123), Parkinson’s disease (44), and spinal cord ischemia-reperfusion injury (124), silencing TREM1 or blocking TREM1 signaling using inhibitory decoy peptides such as LP17, LR12, M3, or GF9 inhibited the release of inflammatory mediators and improved prognosis. Although initially described in bacterial infections, a role for TREM1 during viral infections is emerging. For instance, viral signaling through TLR3 (125) or TLR8 (38) elicits the upregulation of TREM1 in monocytes and macrophages, respectively, and activation of TREM1 in neutrophils synergizes with TLR7 and TLR8 signaling to elicit proinflammatory effector mechanisms (126). A number of viruses, including Ebola virus (127), hepatitis C virus (37), Marburg virus, West Nile virus (128, 129), and Zika virus (130), have also been shown to upregulate the expression and/or activation of TREM1, in many cases leading to the enhancement of the proinflammatory immune response. Furthermore, infection with Crimean-Congo hemorrhagic fever orthonairovirus (131), dengue virus (132), hepatitis B virus (133), hepatitis C virus (133), and West Nile virus (129) all increase the expression and shedding of soluble TREM1 (sTREM1) from infected cell membranes. The role of sTREM1 in the context of viral pathogenesis has yet to be fully elucidated, although the current hypotheses include that it could be a compensatory mechanism to counteract inflammatory processes and attenuate downstream proinflammatory signals. Although combined measurements of cerebrospinal fluid TNFRSF6B (decoy receptor 3) and sTREM1 concentrations can be used to predict nosocomial bacterial meningitis (50), the potential of sTREM1 as a marker of disease severity during viral infections is unknown.

Although thought to be inducible only in response to proinflammatory stimuli, PTGS2 is constitutively expressed within the CNS, where it plays a role in memory and learning, and is upregulated in hippocampal and cortical glutamatergic neurons, where it is involved in long-term synaptic plasticity and functional hyperemia (134, 135). However, as with TREM1, elevated levels of PTGS2 have been described in several neurological disorders, including Alzheimer’s disease, amyotrophic lateral sclerosis, bipolar disorder, Creutzfeldt-Jakob disease, epilepsy, multiple sclerosis, Parkinson’s disease, schizophrenia, and stroke, with PTGS2 inhibitors exhibiting neuroprotective effects (136). PTGES is also significantly elevated in neurons and microglia in Alzheimer’s disease (137) and after transient forebrain ischemia (138), and it may play a pathophysiological role in autoimmune encephalomyelitis (139). Although the role of PTGS2 and PTGES in HIV-associated neurological disorders has still not been elucidated, it is possible that the prostanoids induced by PTGS2 and PTGES are partly responsible for HIV-associated neurological disorders (140). In PLWH, PTGS2-positive macrophages have been detected infiltrating the brains of PLWH with HIV encephalitis (141), and in mice, intracranial infusions of Tat or gp120 induce a PTGS2-mediated proinflammatory response (140, 142, 143). Serum PGE₂ levels are also elevated in PLWH compared with uninfected individuals (144), and increased cerebrospinal fluid PGE₂ levels are positively associated with severity of HIV-associated dementia (145). Furthermore, celecoxib downmodulated the immune activation related to clinical progression of chronic HIV-infection and improved T cell–dependent functions in vivo (146), and an increase in PGE₂ has been shown to contribute to an increase in viral replication in HIV-infected macrophages after the phagocytosis of apoptotic cells (147). Another point to consider is the potential effect of antiretrovirals on prostanoid synthesis. For example, maraviroc activates and increases PTGS2 mRNA expression and PGE₂ release from microglia, a proinflammatory activation that could worsen neurological complications (148). In addition, inhibition of mechanistic target of rapamycin (mTOR) in activated microglia induces autophagy, increases the expression of PTGS2 and PTGES, and increases the release of PGE₂ (149). Tat induces autophagy in microglia (150), and in this study we demonstrate that it also increases the expression of PTGS2 and PTGES and the synthesis of PGE₂ in MMGs.

Although we and others have demonstrated that the in vitro model of HIV infection of microglia used in this study is superior to microglial cell lines on a morphological, transcriptional, and functional level and is similar to ex vivo microglia HIV infection in overall gene, restriction factor, and HIV expression, they are not brain-derived microglia, and there are still some differences. However, the technical challenges of obtaining fresh human brain tissue, the limited number of viable microglia available postisolation, the loss of phenotypic characteristics during the isolation procedure (which continue to deteriorate during ex vivo culture), and further difficulties in performing experiments with primary human brain microglia limited our ability to perform this work using primary microglia. Future work performed using this in vitro model of HIV-infected microglia grown in 3D cerebral organoids to assess the role of TREM1 within the context of the CNS microenvironment and other CNS cell types may provide additional information on the effect HIV has on sTREM1 production. Moreover, because calcineurin/NFAT signaling leads to the downregulation of SLC1A2 glutamate transporters, which can increase extracellular glutamate levels and excitotoxicity at synaptic connections and disrupt astrocyte endfeet and/or blood-brain-barrier integrity, future work should also determine any role Tat may have in disrupting the blood-brain barrier through calcineurin/NFAT signaling activation (151).

In summary, this study demonstrates an important role and mechanism for TREM1 in inhibiting apoptosis of HIV-infected microglia, possibly contributing to viral persistence during HIV infection. Moreover, the present data highlight the potential of TREM1 as a therapeutic target in an HIV cure strategy.

Disclosures

The authors have no financial conflicts of interest.