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. Author manuscript; available in PMC: 2020 Sep 1.
Published in final edited form as: Glia. 2019 May 23;67(9):1719–1729. doi: 10.1002/glia.23642

HIV-1 Tat promotes astrocytic release of CCL2 through MMP/PAR-1 signaling

P Lorenzo Bozzelli 1,2, Tao Yin 2, Valeria Avdoshina 2, Italo Mocchetti 1,2, Katherine E Conant 1,2, Kathleen A Maguire-Zeiss 1,2
PMCID: PMC6640102  NIHMSID: NIHMS1029844  PMID: 31124192

Abstract

The HIV-1 protein Tat is continually released by HIV-infected cells despite effective combination antiretroviral therapies (cART). Tat promotes neurotoxicity through enhanced expression of proinflammatory molecules from resident and infiltrating immune cells. These molecules include matrix metalloproteinases (MMPs), which are pathologically elevated in HIV, and are known to drive CNS injury in varied disease settings. A subset of MMPs can activate G-protein coupled protease-activated receptor 1 (PAR-1), a receptor that is highly expressed on astrocytes. Although PAR-1 expression is increased in HIV-associated neurocognitive disorder (HAND), its role in HAND pathogenesis remains understudied. Herein, we explored Tat’s ability to induce expression of the PAR-1 agonists MMP-3 and MMP-13. We also investigated MMP/PAR-1-mediated release of CCL2, a chemokine that drives CNS entry of HIV infected monocytes and remains a significant correlate of cognitive dysfunction in the era of cART. Tat exposure significantly increased the expression of MMP-3 and MMP-13. These PAR-1 agonists both stimulated the release of astrocytic CCL2, and both genetic knock-out and pharmacological inhibition of PAR-1 reduced CCL2 release. Moreover, in HIV-infected post-mortem brain tissue, within-sample analyses revealed a correlation between levels of PAR-1-activating MMPs, PAR-1, and CCL2. Collectively, these findings identify MMP/PAR-1 signaling to be involved in the release of CCL2, which may underlie Tat-induced neuroinflammation.

Keywords: Matrix metalloproteinase, protease activated receptor 1, Tat, HIV, CCL2

Graphical Abstract

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INTRODUCTION

Approximately 34 million people are currently infected with human immunodeficiency virus (HIV) (Nightingale et al., 2014). Despite combination antiretroviral therapy (cART) having drastically improved life expectancy, up to 50% of HIV+ individuals continue to experience some degree of HIV-associated neurocognitive disorder (HAND) (Heaton et al., 2010; Saylor et al., 2016). The precise mechanisms underlying HAND pathogenesis have yet to be elucidated; however, accumulating evidence has identified the release of soluble factors from astrocytes, microglia, and infiltrating peripheral immune cells to be major contributors.

In the post-cART era, chronic inflammation persists in the HIV+ brain (Tavazzi et al., 2014), which suggests that factors beyond viral replication are involved in HAND. One such factor that exerts cytotoxic effects is the HIV-1 protein transactivator of transcription (Tat), which is released from infected cells (Li et al., 2009). Importantly, it has been shown that cART does not reduce the production of Tat protein, given that it is detected in the cerebrospinal fluid (CSF) of HIV+ patients receiving cART (Johnson et al., 2013). Recent evidence using a Tat transgenic mouse shows that relatively low-level and chronic exposure to Tat can activate astrocytes and induce inflammation (Dickens et al., 2017). Interestingly, anti-Tat antibody titers in CSF are higher in patients without neurocognitive dysfunction than in those patients with HAND, suggesting that Tat itself may be a significant contributor to HAND pathogenesis, and that a Tat-specific immune response may be therapeutically relevant (Bachani et al., 2013).

Similarly, elevated levels of the potent chemokine C-C motif chemokine 2 (CCL2), formerly termed monocyte-chemoattractant protein 1 (MCP-1), continue to persist in the HIV CNS despite cART viral suppression (Kamat et al., 2012; Yuan et al., 2013). CCL2 levels are significantly elevated in the CSF of HIV-dementia patients (Conant et al., 1998), and expression of the CCL2 receptor, CCR2, is increased in HIV-infected monocytes (Williams et al., 2013). Levels of CCL2 have also been shown to correlate with HIV-encephalitis (Cinque et al., 1998). Specifically, Tat protein alone is able to promote CCL2 release from astrocytes (Conant et al., 1998), and monocyte infiltration (Weiss et al., 1999; Pu et al., 2003), which suggests that Tat-mediated CCL2 signaling may reinforce inflammation through the recruitment of immune cells.

We and others have shown that matrix metalloproteinases (MMPs), a major class of enzymes involved in extracellular matrix remodeling, are upregulated in HIV (Conant et al., 1999; Liuzzi et al., 2000; Johnston et al., 2001; Suryadevara et al., 2003; Liuzzi, 2004; Ragin et al., 2009, 2011; Gramegna et al., 2011; Li et al., 2013; Abassi et al., 2017; Xing et al., 2017). HIV-1 Tat induces MMP expression in various cell types (Kumar et al., 1999; Rumbaugh et al., 2006; Ju et al., 2009; Xu et al., 2012; Woollard et al., 2014) promoting neurotoxicity which can be abrogated by MMP inhibition (Johnston et al., 2001). Interestingly, a subset of MMPs, MMP-1, −3, and −13, are able to activate the inflammation-associated protease-activated receptor 1 (PAR-1) (Boire et al., 2005; Tressel et al., 2011; Jaffré et al., 2012; Brzdak et al., 2017). Importantly, PAR-1 expression in astrocytes is elevated in the HIV encephalitis brain (Boven et al., 2003; Kim et al., 2015b). Furthermore, gene expression of a PAR-1-activating MMP has been found to be significantly dysregulated in HIV-infected brains when compared to non-infected brains (Sanna et al., 2017).

Important to our studies, Tat alone is able to induce astrogliosis (Zhou et al., 2004; Ton and Xiong, 2013; Fan and He, 2016a; b) and inhibit astrocyte Wnt/β-catenin signaling (Henderson et al., 2012) which modulates HIV replication in peripheral monocytes (Richards et al., 2015). While PAR-1 is expressed in many cell types, its expression is particularly enriched in astrocytes (Junge et al., 2004). Also, given that PAR-1 activation in non-neural cells promotes CCL2 release (Wang et al., 2007), and that astrocytes are known to express and release this chemokine, we explored Tat-relevant PAR-1/chemokine signaling in astrocytes. Here, we report that Tat induction of astrocytic CCL2 is significantly attenuated by MMP inhibition. In examining Tat-induced MMPs, we found that Tat increased levels of MMP-3 and MMP-13, and that these PAR-1-activating MMPs stimulate CCL2 release. Genetic knock-out or pharmacological inhibition of PAR-1 reduces MMP-induced CCL2 release. Furthermore, within-sample analyses of individual HIV-infected post-mortem brain tissues revealed that levels of MMP-3 and −13 were both significantly correlated with CCL2. These results identify MMP/PAR-1 signaling as a novel pathway whereby the cART-resistant Tat protein may be reinforcing HAND.

MATERIALS AND METHODS

Reagents

HIV-1 IIIB Tat recombinant protein was obtained through the AIDS Reagent Program, Division of AIDS, NIAID, NIH (Catalog # 2222). To protect against oxidation, Tat recombinant protein was reconstituted in PBS containing 0.5 mM DTT (dithiothreitol). Tat was used at a final concentration of 100 ng/mL and the DTT-containing reconstitution buffer served as control in experiments involving Tat. To rule out endotoxin contamination, Tat was heat-inactivated at 95° C for 30 minutes (data not shown). The broad-spectrum MMP inhibitor GM6001 (Tocris, Catalog # 2983) was reconstituted in DMSO (dimethyl sulfoxide) and used at a final concentration of 10 µM. Human recombinant pro-MMP-13 (R&D Systems, Catalog # 511-MM-010) and human recombinant catalytic MMP-13 (cMMP-13; Enzo Life Sciences, Catalog # BML-SE246–0010) were both used at a final concentration of 20 nM. Dose-curve experiments revealed that 20 nM of cMMP-13 resulted in maximal CCL2 release (data not shown). Human recombinant catalytic MMP-3 (cMMP-3; Millipore Calbiochem, Catalog # 444217) was used at a final concentration of 4.5 nM. Mouse recombinant IL-1β (R&D Systems, Catalog # 401-ML-CF) was used at 100 ng/mL. PBS was used as control in experiments involving IL-1β, pro-MMP-13, catalytic MMP-3, and catalytic MMP-13. Vorapaxar (Axon Medchem, Catalog # 1755) was reconstituted in DMSO and used at a final concentration of 2 µM.

Primary astrocyte culture

Primary astrocyte cultures were derived from the cortices of C57BL/6 or PAR-1 knockout (PAR1-KO) (The Jackson Laboratory, stock # 002862) neonatal pups of both sex and prepared as previously described (Daniele et al., 2014). More specifically, brains were isolated from postnatal day 2–3 mice and meninges removed. Cortices were microdissected and minced in medium (HBSS supplemented with 100 μg/mL penicillin/streptomycin & 0.01 M HEPES). Minced tissue was centrifuged (1000 RPM; 5 min; RT), resuspended in complete medium (Minimal Essential Medium Earle’s [MEM] supplemented with: 1 mM L-glutamine, 1 mM sodium pyruvate, 0.6% v/v D-(+)-glucose, 100 μg/mL penicillin/streptomycin (P/S), 4% v/v fetal bovine serum (FBS), 6% v/v horse serum) and cultured in T-75 flasks in a humidified tissue culture incubator (37°C, 5% CO2). Astrocyte purification was completed on DIV 15 – 20, whereby flasks were placed on a rotary shaker initially for four hours (200 RPM, 37°C) to detach unwanted cells. Media was removed and replaced with fresh medium followed by shaking overnight. The following day, media was removed, and adherent astrocytes were detached using 1X trypsin/EDTA for 5 minutes at 37° C. Astrocytes were passaged into a new flask and cultured in astrocyte medium (MEM supplemented with 10% FBS, 1% P/S and 1 mM L-glutamine). Astrocytes were shaken as mentioned above prior to any subsequent passages. Astrocytes used for experiments were passaged 2 – 5 times. Experiments were conducted at DIV 18 – 53 under serum-free conditions. The duration of culture treatments is mentioned in the figure captions below. All procedures were approved by Institutional Animal Care and Use Committee at Georgetown University and conducted following guidelines for animal experimentation.

MMP-13 activity assay

Following treatment, astrocyte conditioned media was collected and stored at −80°C until use. MMP-13 activity was measured in conditioned media using the SensoLyte Plus 520 MMP-13 Assay Kit (Anaspec, Catalog # AS-72019) as per manufacturer’s instructions with the exception of steps involving immunodetection and washes. End-point fluorescent intensity was measured at excitation/emission wavelength = 485 nm/535 nm. In order to determine levels of total MMP-13, astrocyte conditioned media samples were incubated with APMA (4-aminophenylmercuric acetate), which activates matrix metalloproteinases.

ELISAs

Secreted protein levels in astrocyte-conditioned media were assessed using the CCL2/MCP-1 (R&D Systems, Catalog # MJE00) and IL-6 (R&D Systems, Catalog # M600B) enzyme-linked immunosorbent assays (ELISAs) according to the manufacturer’s instructions.

Quantitative real-time PCR

Total cellular RNA was isolated from treated astrocytes using the RNAeasy kit (QIAGEN, Catalog # 74106) according to the manufacturer’s instructions. The procedure utilized on-column DNA digestion with DNAse (QIAGEN, Catalog # 79254). RNA concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Equal amounts of total RNA from all samples were reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Catalog # 4368814). Real-time quantitative PCR was subsequently performed using TaqMan Universal PCR Master Mix and a 7900HT Fast Real-time PCR System. The gene specific primers were: MMP-13 (Applied Biosystems, Mm00439491_m1) and 18s rRNA (Applied Biosystems, Hs99999901_s1). Data were analyzed using the relative quantification ΔΔCt method with normalization of target gene expression to the internal control 18s rRNA, followed by normalization to control samples. Gene expression changes are represented as fold change (2-ΔΔCt). Statistical analyses were conducted on the fold change values.

Western blot

Cortical tissue was microdissected from post-mortem HIV-infected human brains provided by the National NeuroAIDS Tissue Consortium and homogenized in RIPA lysis buffer with protease/phosphatase inhibitors. Protein concentrations were determined using the BCA protein assay (Pierce) and samples were mixed with 2X Laemmli buffer containing β-mercaptoethanol and heated at 95° C for 5 minutes. 20 µg of total protein were loaded per lane and resolved in a 4% - 20% gradient polyacrylamide tris glycine gel (BioRad). Following electrophoresis, proteins were transferred to nitrocellulose membrane and blocked with 1X TBS-Tween20 (TBS-T) containing 5% non-fat milk. Membranes were then incubated overnight at 4° C with the following primary antibodies diluted in blocking buffer: mouse anti-MMP-13 (ThermoFisher, MA5–14238, 1:100); goat anti-CCL2/MCP-1 (R&D Systems, AF-279-NA, 1:1000); Rabbit anti-Thrombin R (PAR-1; Santa Cruz Biotechnology, SC-5605, 1:1000); mouse anti-GAPDH (Millipore, MAB374, 1:1000); rabbit anti-MMP-3 (Abcam, ab52915, 1:2000). Afterwards, membranes were washed multiple times with 1X TBS-T, incubated at room temperature with HRP-conjugated secondary antibodies, followed by additional washes prior to signal detection using Pierce ECL substrate (ThermoFisher). Following CCL2/MCP-1 and GAPDH immunodetection, membranes were stripped using a mild stripping buffer, developed with ECL to ensure sufficient removal of prior secondary antibodies, re-blocked, and probed with an anti-MMP-3 antibody as mentioned above. Images were acquired using the GE Amersham Imager 600 and bands were quantified using ImageJ (NIH). Band densities of MMP-13, CCL2, PAR-1, and MMP-3 were normalized to GAPDH.

Statistical analyses

All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc.). Data were subjected to unpaired t-test or one-way ANOVA with multiple comparison tests as noted. Pearson correlation coefficients were calculated for relationships between protein levels in human brain tissue. All data are reported as mean ± SEM and significance was set at p ≤ 0.05. Experiments were repeated 1 – 4 times using separate astrocyte cultures. Statistical analyses were conducted using biological replicates which reflect averages of technical replicates taken from the separate cultures (see figure legends for details).

RESULTS

Tat-induced CCL2 release is significantly reduced by inhibition of MMP activity

Tat has been shown to potently stimulate CCL2 release (Conant et al., 1998; Toborek et al., 2003; El-Hage et al., 2005) which we also observe following 24-hour exposure of mouse astrocytes to 100 ng/mL of recombinant Tat (unpaired t test, df = 4, t = 3.881, p = 0.0178; Figure 1A). Tat has also been shown to increase MMP expression (Kumar et al., 1999; Johnston et al., 2001) which led us to evaluate whether Tat-induced CCL2 release may be MMP-dependent. Astrocytes were exposed to Tat ± the broad-spectrum MMP inhibitor, GM6001. Addition of GM6001 led to a significant reduction in Tat-induced CCL2 release (one-way ANOVA, F(3,4) = 19.94, p = 0.0072; Newman-Keuls multiple comparisons test: Tat vs. Tat + GM6001, p < 0.05; Figure 1B). These results suggest that a significant amount of Tat-induced CCL2 release is dependent upon MMP activity.

Figure 1.

Figure 1.

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Tat-induced CCL2 release from astrocytes is significantly reduced by MMP inhibition. A. Primary mouse cortical astrocytes were treated for 24-hours under serum-free conditions. Treatment with 100 ng/mL of recombinant Tat promoted CCL2 release as detected by ELISA. Unpaired t test, p < 0.0178, N = 3 biological replicates (3 separate cultures with 4 - 6 replicates per condition in each culture). B. Cells were treated the same as the previous experiment with or without the broad-spectrum MMP-inhibitor GM6001 (10 µM). GM6001 significantly attenuated Tat-induced CCL2 release. One-way ANOVA followed by Newman-Keuls multiple comparisons test, p < 0.05, N = 2 biological replicates (2 separate cultures with 3 replicates per condition in each culture). * p < 0.05

Tat increases levels of the PAR-1 agonists MMP-3 and MMP-13

MMP-3 and −13 are two known MMPs that are able to activate PAR-1. Tat exposure resulted in a significant increase of MMP-3 protein in astrocyte cultures (unpaired t test, df = 4, t = 7.572, p = 0.0016; Figure 2A). Notably, MMP-13 expression has been identified as a particularly altered MMP in the HIV-infected brain (Sanna et al., 2017); therefore, we focused the majority of subsequent experiments on MMP-13. Quantitative RT-PCR revealed Tat exposure significantly elevated MMP-13 mRNA fold expression (unpaired t test, df = 4, t = 10.45, p < 0.0005; Figure 2B). An activity assay was also performed to determine whether conditioned media from control and Tat-stimulated astrocytes contained potentially active enzyme species. Results are shown in Figure 2C and confirm the presence of such species.

Figure 2.

Figure 2.

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Tat induces MMP-3 and MMP-13 expression in astrocyte cultures. A. Astrocytes were treated as described in the previous experiments. Tat exposure led to an increase in MMP-3 protein levels as detected by ELISA. Unpaired t test, p = 0.0016, N = 3 biological replicates (3 separate cultures with 3 - 6 replicates per condition in each culture). B. MMP-13 mRNA was increased by Tat when compared to control. Unpaired t test, p = 0.0005, N = 3 biological replicates (3 separate cultures with 6 replicates per condition in each culture); mRNA fold expression normalized to controls with statistical analysis done using the fold change values. C. A specific MMP-13 activity assay was used to detect potentially active enzyme species in the conditioned media of control and Tat-stimulated astrocyte cultures. Unpaired t test, p = 0.1709, N = 2 biological replicates (2 separate cultures with 6 replicates per condition in each culture). ** p < 0.01, *** p < 0.001

PAR-1-activating MMPs promotes CCL2 release via PAR-1

PAR-1 expression is increased in astrocytes during HIV encephalitis (Boven et al., 2003); however, the contribution of increased PAR-1 expression in HAND pathogenesis remains largely unknown. PAR-1 is generally considered to be an inflammation-associated receptor (Noorbakhsh et al., 2003) and activation of PAR-1 in non-neural cells results in the release of CCL2 (Wang et al., 2007). As a potent agonist of PAR-1, we sought to determine the ability of MMP-13 to promote CCL2 release. We compared the following peptides for their ability to stimulate CCL2 release: the pro-inflammatory cytokine IL-1β, pro-MMP-13, and catalytic MMP-13. IL-1β and catalytic MMP-13 similarly stimulated a large release of CCL2 (Figure 3A). However, Pro-MMP-13 treatment did not appear to differ from control. This latter observation may indicate that longer incubations are necessary for the conversion of pro-enzyme to an active form.

Figure 3.

Figure 3.

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PAR-1 activating MMPs induce CCL2 release from astrocytes. A. Astrocytes were treated for 24-hours with pro-MMP-13, catalytic MMP-13 (cMMP-13) and IL-1β. Catalytic MMP-13 was comparable to IL-1β in stimulating CCL2 release, while pro-MMP-13 was similar to control. Experiment conducted two times with separate cultures; single experiment shown with 3 technical replicates per condition. Control levels were non-detectable (N.D.); B. Catalytic MMP-13 also stimulated release of the pro-inflammatory cytokine IL-6. Unpaired t test, p = 0.0098, N = 3 biological replicates (3 separate cultures with 4 replicates per condition in each culture). C. cMMP-3 was also found to potently stimulate CCL2 release. Unpaired t test, p < 0.0001, N = 4 biological replicates (4 separate cultures with 3 - 4 replicates per condition). ** p < 0.01, **** p < 0.0001

In addition to CCL2, Tat has also been shown to promote release of IL-6 (Nath et al., 1999; Nookala and Kumar, 2014), a pro-inflammatory molecule implicated in HIV pathogenesis (Perrella et al., 1992). We therefore evaluated IL-6 levels in astrocyte cultures exposed to recombinant catalytic MMP-13. We observed that cMMP-13 significantly increased IL-6 levels (unpaired t test, df = 4, t = 4.637, p = 0.0098; Figure 3B). Additionally, we examined whether another PAR-1 agonist, MMP-3, was able to similarly stimulate CCL2 release. We observed that cMMP-3 exposure elevated CCL2 release from cultured astrocytes (unpaired t test, df = 6, t = 12.05, p < 0.0001; Figure 3C). Together, these data suggest a role for Tat-induced PAR-1-activating MMPs in promoting both cytokine and chemokine release.

Both genetic KO and blockade of PAR-1 reduce CCL2 release

Subsequent astrocyte cultures were derived from wildtype (WT) and PAR-1 KO mice. We first assessed the potential for astrocytes derived from both genotypes to release CCL2 by treating cells with the HIV-relevant pro-inflammatory cytokine IL-1β. WT and PAR-1 KO astrocytes did not differ in their release of IL-1β-induced CCL2 release (unpaired t test, df = 4, t = 0.1764, p = 0.8686; Figure 4A). We did however observe that PAR-1 KO astrocytes displayed a significant reduction in the amount of MMP-13-induced CCL2 release (unpaired t test, df = 4, t = 14.31, p = 0.0001; Figure 4B), suggesting that PAR-1 is a major signaling pathway involved in MMP-induced CCL2 release.

Figure 4.

Figure 4.

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Genetic KO or blockade of PAR-1 reduces CCL2 release following MMP exposure. A. Primary cortical astrocytes were derived from WT or PAR-1 KO mice and treated with IL-1β. WT and PAR-1 KO astrocytes did not differ in their ability to release elevated CCL2 in response to IL-1β stimulation. Unpaired t test, p = 0.8686, N = 3 biological replicates (3 separate cultures with 3 - 4 replicates per condition in each culture). B. Astrocytes from both genotypes were treated with 20 nM of cMMP-13. cMMP-13 treated PAR-1-KO astrocytes displayed a reduction in CCL2 release when compared to WT astrocytes. Unpaired t test, p = 0.0001, N = 3 biological replicates (3 separate cultures with 3 - 4 replicates per condition in each culture). C. Pharmacological inhibition of PAR-1, using 2 µM vorapaxar, resulted in a significant reduction in MMP-induced CCL2 release. Unpaired t test, p = 0.0135, N = 3 biological replicates (3 separate cultures with 4 replicates per condition in each culture). * p < 0.05, *** p = 0.0001

We then repeated the same experiment as above in WT astrocytes, and in the presence or absence of the FDA-approved PAR-1 antagonist vorapaxar. Similar to the PAR-1 KO findings, pharmacological blockade of PAR-1 resulted in a significant reduction in MMP-induced CCL2 release (unpaired t test, df = 4, t = 4.220, p = 0.0135; Figure 4C). These data identify a role for astrocytic PAR-1 signaling in MMP-induced CCL2 release.

PAR-1-activating MMPs correlate with CCL2 in HIV-infected human brain tissue

Though previous work has indicated that levels of MMP-13 mRNA correlate with cognitive dysfunction in HIV infected individuals, protein levels have not been assessed. An in vivo relationship between PAR-1-activating MMPs and CCL2 has also not been demonstrated. We investigated the relative amounts of PAR-1-activating MMPs and their relationship with both PAR-1 and CCL2 in HIV-infected post-mortem human brain tissue by Western blot analysis (Table 1). MMP-3 and a band corresponding with the catalytic domain of MMP-13 were readily detected in tissue samples (Figure 5A and 5B). Given the heterogeneity inherent to human clinical samples and the small sample size, we evaluated within-sample associations and observed that the amount of cMMP-13 was significantly correlated with both CCL2 (Pearson correlation r = 0.6581, r2 = 0.4332, p = 0.0277, n = 11; Figure 5C) and the receptor-substrate PAR-1 (Pearson correlation r = 0.8019, r2 = 0.6431, p = 0.003, n = 11; Figure 5C). The amount of MMP-3 was also significantly correlated with CCL2 (Pearson correlation r = 0.9247, r2 = 0.8551, p < 0.0001, n = 11; Figure 5C). These data present a correlative relationship between levels of PAR-1-activating MMPs and CCL2.

Table 1.

Post-mortem brain tissue sample information.

Age Sex Race Exposure Viral load CD4 count
40 F White Heterosexual 77 170
49 M White IV drug user undetectable 103
60* M Black IV drug user undetectable 38
46 F Native Alaskan/American Indian Heterosexual >750000 5
34 M White Unknown 54148 unknown
42 M Hispanic Homosexual 162642 16
51 F Black IV drug user 20103 198
46 M White Homosexual undetectable 233
51 M White IV drug user/ homosexual 1000000 30
44 F Native Alaskan/American Indian Heterosexual >750000 66
49 M Native Alaskan/American Indian, White Homosexual <400 1
50 M White Homosexual 303640 11
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*

outlier sample excluded from statistical analysis

Figure 5.

Figure 5.

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PAR-1-activating MMPs are detected in HIV+ post-mortem brain tissue and MMP levels correlate with CCL2. A. Representative Western blots shown for MMP-13 and PAR-1. In addition to the pro- and cleaved forms of MMP-13, a prominent band corresponding to the active catalytic domain of MMP-13 (~25 kDa) was readily detected. The PAR-1 antibody revealed bands corresponding to glycosylated and unglycosylated forms (66 and 47 kDa, respectively). Data were normalized to GAPDH. B. Representative Western blots shown for CCL2 and MMP-3. The CCL2 antibody revealed multiple bands corresponding to glycosylated forms of the protein. A single band was detected for MMP-3 (~54 kDa). Data were normalized to GAPDH. Each vertical lane across the representative blots corresponds to the same human sample. C. Within individual samples, protein levels were positively correlated. The catalytic domain of MMP-13 was significantly correlated with PAR-1 (Pearson correlation; r = 0.80, r2 = 0.64, p = 0.003, n = 11), and with CCL2 (Pearson correlation; r = 0.66, r2 = 0.43, p = 0.028, n = 11). MMP-3 was significantly correlated with CCL2 (Pearson correlation; r = 0.92, r2 = 0.86, p < 0.0001, n = 11). Best fit line and 95% confidence band generated using linear regression.

DISCUSSION

Tat has been shown to promote the release of various MMPs from different cell types (Kumar et al., 1999; Johnston et al., 2001; Conant et al., 2004; Ju et al., 2009; Xu et al., 2012; Woollard et al., 2014); however, the ability of Tat to induce PAR-1-activating MMPs has remained largely unexplored. We found that Tat induced MMP-3 and −13 expression in astrocyte cultures. Given that PAR-1 is an MMP substrate abundantly expressed on astrocytes (Junge et al., 2004; Allen et al., 2016), we examined whether MMP-induced CCL2 release occurred via PAR-1. Both genetic KO and pharmacological inhibition reduced MMP induction of CCL2, which provides evidence for this signaling pathway in chemokine release. Here we propose a model whereby Tat is able to promote MMP/PAR-1 chemokine signaling and result in subsequent monocyte recruitment (Figure 6).

Figure 6.

Figure 6.

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Hypothetical model illustrating Tat-induced CCL2 release via MMP/PAR-1 signaling. Recruitment of monocytes via CCL2 signaling may potentiate neuroinflammation that is characteristic of the HIV-infected brain. These pathological signaling pathways may underlie symptoms associated with HAND.

Macrophage/monocyte lineage cells are major contributors to HIV pathogenesis (Garden, 2002; Williams et al., 2014), and Tat has been shown to stimulate CCL2 release from astrocytes (Conant et al., 1998; Weiss et al., 1999; El-Hage et al., 2005), which promotes monocyte invasion into the CNS (Pu et al., 2003). CSF CCL2 is increased in HIV-dementia patients and correlates with severity of dementia (Kelder et al., 1998). Plasma CCL2 is a reliable predictive biomarker in HAND pathogenesis (Ragin et al., 2010) and correlates with brain injury (Ragin et al., 2006). Elevated blood levels of monocyte activation markers are correlated with worse cognitive performance in HIV-infected individuals despite suppression of plasma HIV RNA. This evidence supports the notion that impaired cognition may be a consequence of other factors beyond viral replication (Imp et al., 2017).

Additionally, inflammation/gliosis can occur independently of cell infection whereby the release of soluble viral proteins from infected cells activates non-infected cells, and promotes pro-inflammatory cytokine and chemokine release. This is of major clinical importance given that CNS inflammation persists even when HIV replication is suppressed by cART (Yilmaz et al., 2008; Dahl et al., 2011; Johnson et al., 2013). Tat is thought to play a major role in this pro-inflammatory state in that levels of Tat are refractory to cART and likely continue to promote cytotoxicity despite reduced viral replication (Johnson et al., 2013). Furthermore, Tat induces IL-1β production in both monocytes (Nath et al., 1999; Yang et al., 2010) and microglia (Chivero et al., 2017); therefore, it is possible that Tat induction of CCL2 and the ensuing recruitment of monocytic cells results in the release of pro-inflammatory cytokines and MMPs which likely reinforce a pathological cycle (Figure 6).

In support of the proposed cyclical model, previous work had found that genetic knock-out of CCL2 resulted in a decrease of MMP-13 (Raghu et al., 2017). Furthermore, this signaling pathway is likely to promote progressive inflammation given that the inflammatory molecules IL-1β and IL-6 have been shown to upregulate PAR-1 (Acharjee et al., 2011; Lee et al., 2017). Relevant to PAR-1 as a therapeutic target, the FDA has recently approved the PAR-1 antagonist vorapaxar as an antiplatelet therapy (Fala, 2015). We found that vorapaxar decreased MMP-induced CCL2 release. These findings suggest that targeting PAR-1 may be beneficial in conditions whereby MMP activity is aberrantly elevated. As such, PAR-1 targeted therapies may be able to block progressive inflammatory cycles.

Interestingly, CCL2 has also been shown to protect neurons against Tat-induced toxicity, highlighting a paradoxical role for this chemokine in the context of HIV infection (Eugenin et al., 2003; Li et al., 2009). Further evidence for the dual role of CCL2 in HIV comes from findings in individuals with genetic variants that promote CCL2 expression whereby carriers of this variant are at a lower risk of acquiring HIV-1; however, once infected, the same genetic variant promotes monocyte infiltration, increases risk for HIV-associated dementia (Gonzalez et al., 2002), and is associated with increased levels of pro-inflammatory molecules in CSF (Thames et al., 2015). Relevant to our findings, a recent clinical trial of virologically-suppressed patients treated with cenicriviroc, a dual CCR2/CCR5 chemokine receptor antagonist, revealed an improvement in cognitive performance (DʼAntoni et al., 2018). Cenicriviroc has been shown to be effective in reducing transmigration of monocytes, and as such may be useful in reducing infiltration of infected monocytes into the CNS (Veenstra et al., 2017).

In terms of MMP proteolytic activity, recent work has shown that the soluble cleavage product of intercellular adhesion molecule-5 (ICAM-5), an MMP substrate, is elevated in CSF and plasma of HIV+ patients with impaired cognition compared to those without cognitive impairment (Yuan et al., 2017). The increased levels of MMP substrate cleavage fragments provide evidence for a role of aberrant MMP activity in the context of impaired cognition. We have previously shown that the MMP-cleaved ectodomain of the adhesion molecule N-cadherin promotes microglial activation (Conant et al., 2017) which may reflect an alternate pathway whereby HIV promotes MMP-dependent gliosis.

An extensive body of literature has found correlations between various MMPs, brain atrophy, and HAND diagnosis (Conant et al., 1999; Liuzzi et al., 2000; Ragin et al., 2009, 2011; Li et al., 2013; Xing et al., 2017). Inhibitors of the MMP substrate PAR-1 protect against MMP-mediated damage of the blood-brain barrier (Kim et al., 2015a), a prominent pathology in the HIV brain (Atluri et al., 2015), suggesting additional possible roles for MMP/PAR-1 inhibition in HIV infection. Of note, our findings do not rule out the contribution of other MMPs released from additional cell types in response to Tat given that we used a broad-spectrum MMP inhibitor in our experiments. Future experiments will be needed to expand upon the Tat-induced MMP response as well as that of other inducible agonists of PAR-1.

Within individual post-mortem HIV-infected brain samples, we observed a correlation between levels of MMPs and CCL2. Major limitations exist in the interpretation of such analyses given that only within-subject correlations were possible given the inherent heterogeneity of human tissue and the small sample size. A larger sample size would be necessary in order to assess a correlation between PAR-1-activating MMP levels and clinical diagnosis. Future studies should assess these MMPs in cerebrospinal fluid and/or blood as a potential biomarker of HAND severity and/or clinical progression.

Taken together, the data presented here identify a novel mechanism whereby Tat may promote CCL2 release and propagate HIV neurotoxicity. Our proposed model, whereby soluble HIV protein promotes MMP/PAR-1 signaling of a potent chemokine involved in monocyte recruitment, suggests that further exploration of MMP/PAR-1 signaling is warranted as a possible therapeutic target in HAND.

MAIN POINTS.

HIV Tat-induced matrix metalloproteinases activate protease-activated receptor 1 (PAR-1) and stimulate release of CCL2—a chemokine associated with cognitive impairment. Blockade of PAR-1 reduced CCL2 release; therefore, the MMP/PAR-1 axis may be a therapeutic target in HIV infection.

ACKNOWLEDGMENTS

This study was funded by National Institutes of Health (NIH) R01 NS083410 and R01 NS108810 (KC and KMZ), NIH RO1 NS079172 (IM), NIH T32 NS041218 (PLB), the Georgetown University Medical Campus Graduate Student Research Grant (PLB), and the Cosmos Club Foundation of Washington, D.C. (PLB). Human brain samples were provided by the National NeuroAIDS Tissue Consortium (NNTC) and made possible through NIH funding U24MH100931, U24MH100928, and U24MH100925 to the Manhattan HIV Brain Bank, California NeuroAIDS Tissue Network, and Data Coordinating Center, respectively.

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