IFN-γ Mediates Enhancement of HIV Replication in Astrocytes by Inducing an Antagonist of the β-Catenin Pathway (DKK1) in a STAT 3-Dependent Manner This information is current as of September 24, 2021. Wei Li, Lisa J. Henderson, Eugene O. Major and Lena Al-Harthi J Immunol 2011; 186:6771-6778; Prepublished online 11 May 2011; doi: 10.4049/jimmunol.1100099 Downloaded from http://www.jimmunol.org/content/186/12/6771

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IFN-g Mediates Enhancement of HIV Replication in Astrocytes by Inducing an Antagonist of the b-Catenin Pathway (DKK1) in a STAT 3-Dependent Manner

Wei Li,*,1 Lisa J. Henderson,* Eugene O. Major,† and Lena Al-Harthi*

Typically, IFN-g is an antiviral cytokine that inhibits the replication of many viruses, including HIV. However, in the CNS, IFN-g induces HIV-productive replication in astrocytes. Although astrocytes in vitro are refractory to HIV replication, recent in vivo evidence demonstrated that astrocytes are infected by HIV, and their degree of infection is correlated with proximity to activated macrophages/microglia. The ability of IFN-g to induce HIV replication in astrocytes suggests that the environmental milieu is critical in regulating the permissiveness of astrocytes to HIV infection. We evaluated the mechanism by which IFN-g relieves restricted HIV replication in astrocytes. We demonstrate that although astrocytes have robust endogenous b-catenin signaling, Downloaded from a pathway that is a potent inhibitor of HIV replication, IFN-g diminished b-catenin signaling in astrocytes by 40%, as evaluated by both active b-catenin expression and b-catenin-mediated T cell factor/lymphoid enhancer reporter (TOPflash) activity. Further, IFN-g–mediated inhibition of b-catenin signaling was dependent on its ability to induce an antagonist of the b-catenin signaling pathway, -related protein 1, in a STAT 3-dependent manner. Inhibition of STAT3 and Dickkopf-related protein 1 abrogated the ability of IFN-g to enhance HIV replication in astrocytes. These data demonstrated that IFN-g induces HIV replication in astrocytes by antagonizing the b-catenin pathway. To our knowledge, this is the first report to point to an intricate http://www.jimmunol.org/ cross-talk between IFN-g signaling and b-catenin signaling that may have biologic and virologic effects on HIV outcome in the CNS, as well as on broader processes where the two pathways interface. The Journal of Immunology, 2011, 186: 6771–6778.

nterferon-g (IFN-g), a type II IFN, is a pleiotropic cytokine neuroinvasion and severity of neuropathogenesis in the human involved in antimicrobial and antitumor immunity by en- brain and the brain of SIV-infected macaques (4, 5). I hancing Ag presentation through MHC class I and class II, The majority of IFN-g effects are mediated by signaling through regulating a variety of , and facilitating proapoptotic re- the JAK–STAT pathway (6). IFN-g signaling through JAK–STAT sponses of infected cells (1). Although IFN-g is predominantly involves an initial step of IFN-g binding to its receptor, leading secreted by NK and NK T cells to activate macrophages and by to oligomerization of the IFN-g receptor subunits (IFNGR1 and by guest on September 24, 2021 effector CD4+ and CD8+ Ag-specific T cells, it is also secreted by IFNGR2), which causes phosphorylation and activation of JAKs. activated astrocytes and microglia in response to mechanical or JAK activation leads to phosphorylation and subsequent activa- ischemic injury (2). Further, IFN-g causes alteration in Ca2+ tion of STAT, which dimerize and translocate to the nucleus, waves in the astrocytic network, which is a marker of astrocyte where they bind g-activated sequences in the promoter of IFN-g– activation and may be important in the formation of synapses (3). regulated genes and, with cooperation from other transcriptional Although IFN-g is associated with enhanced anti-HIV immunity factors, such as breast cancer susceptibility 1 (BRCA1) and in the systemic compartment, in the CNS it is associated with HIV mini- maintenance protein 5 (MCM5), regulate IFN-g–responsive genes. Approximately 500 genes are regulated through the IFN-g–induced JAK–STAT pathway, including IFN- *Department of Immunology/Microbiology, Rush University Medical Center, Chi- inducible protein 10, GTPase, and suppressor of cytokine signal- cago, IL 60612; and †National Institute of Neurological Disease and Stroke, National ing I (1, 6). Seven STAT family members have been identified. Institutes of Health, Bethesda, MD 20892 STAT 3, in particular, is evident in reactive astrocytes and is linked 1 Current address: Department of Infectious Diseases, Beijing You’an Hospital, Cap- to neuroinflammatory responses in rodent models of ischemia ital Medical University, Beijing, China. and spinal cord injuries (7, 8). STAT 3 is activated by cytokines Received for publication January 14, 2011. Accepted for publication April 14, 2011. (IFN-g, IL-6, G-CSF) and growth hormones. It induces cell cycle This work was supported by Grants R01 NS060632 (to L.A.-H.) and F31 NS071999 progression, prevents apoptosis, and may be linked to oncogenesis (to L.J.H.) from the National Institutes of Health. It was also supported by the Chicago Developmental Center for AIDS Research (P30 AI 082151), a National through induction of proto-oncogenes, such as c-myc (9). Institutes of Health-funded program supported by the National Institute of Allergy HIV invades the brain early in the course of disease and leads and Infectious Diseases; the National Cancer Institute; the National Institute of Mental Health; the National Institute of Drug Abuse; the National Institute of Child to progressive neurologic impairments. Prior to the era of highly Health and Development; the National Heart, Lung, and Blood Institute; and the active antiretroviral therapy, HIV led to frank dementia/encepha- National Center for Complementary and Alternative Medicine. litis in ∼25% of HIV-infected individuals. Today, HIV causes Address correspondence and reprint requests to Dr. Lena Al-Harthi, Department of a milder, but much wider, spectrum of neurologic impairments, Immunology and Microbiology, Rush University Medical Center, 1735 West Harri- son Street, 614 Cohn, Chicago, IL 60612. E-mail address: [email protected] described as HIV-associated neurocognitive disorders (HAND). HAND symptoms include memory impairment, depression, Abbreviations used in this article: DN, dominant negative; FLUD, fludarabine; G3I, glycogen synthase kinase-3b inhibitor; GSK3b, glycogen synthase kinase-3b; HAD, tremors, psychosis, seizures, and behavioral changes, to name a HIV-associated dementia; HAND, HIV-associated neurocognitive disorders; HFA, few. Recent assessments from the CNS HIV Antiretroviral Ther- human fetal astrocyte; LEF, lymphoid enhancer; NIH, National Institutes of Health; PDA, progenitor-derived astrocyte; PFA, primal fetal astrocyte; S3I, STAT3 inhibitor; apy Effects Research (CHARTER) study (10) indicated that TCF, T cell factor. HAND occurs in 53% of HIV-infected individuals. HIV-mediated www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100099 6772 INTERACTION BETWEEN IFN-g AND b-CATENIN SIGNALING IN CNS neuropathogenesis, depending on the severity of disease, includes Materials and Methods reactive astrocytosis, myelin pallor, and perturbations in synaptic Generation of primary human fetal astrocytes and human and dendritic density that may also include selective neuronal progenitor-derived astrocytes loss. The mechanism of HIV-mediated neurologic disorder is not Human fetal astrocytes (HFA), isolated at ∼20 wk gestation, were pur- entirely clear, but it is likely driven by both direct (active viral chased from Lonza (BioWhittaker, Walkersville, MD). Progenitor-derived replication) and indirect sequelae to HIV invasion of the brain. astrocytes (PDA) were generated from neural progenitor cells, as pre- Indirect mechanisms include dysregulation of glia, release of viral viously described (32). Briefly, progenitor cells were provided by Dr. , and elevation of neurotoxic proteins (TNF-a, IL-6, IL- Eugene Major (National Institute of Neurological Disorders and Stroke, 1b, TGF-b, endothelin, glutamate) from resident brain cells and National Institutes of Health [NIH]) and seeded on poly-D-lysine–coated T-75 tissue culture flasks at 2 3 106 cells/flask. Cells were maintained infiltrating lymphocytes (11). in progenitor medium consisting of neurobasal media (Life Technolo- The primary targets of HIV infection in the CNS are infiltrating gies Invitrogen, Carlsbad, CA) supplemented with 0.5% bovine albumin monocytes/macrophages and microglia. Astrocytes constitute 40– (Sigma, St. Louis, MO), neurosurvival factor (Lonza), N2 components 60% of brain cells and provide vital functions for brain homeo- (Life Technologies Invitrogen), 25 ng/ml fibroblast growth factor, 20 ng/ml epidermal growth factor (R&D Systems, Minneapolis MN), 50 mg/ml stasis, such as regulation of neuronal development, maintenance gentamicin (Lonza) and 2 mM L-glutamine (Life Technologies Invi- of the blood–brain barrier, metabolism of neurotransmitters, se- trogen). To induce differentiation, progenitor medium was replaced with cretion of neurotrophic factors, and immune surveillance in the PDA medium containing DMEM (Life Technologies Invitrogen) supple- brain by secretion of cytokines/chemokines (12–14). Astrocytes mented with 10% heat-inactivated FBS (Sigma), 2 mM L-glutamine, and . are CD42 but may express alternative receptors for HIV entry, 50 mg/ml gentamicin. Cultures were 90% positive for GFAP after 30 d of differentiation. HFA refer to HFA or PDA. including D6, a promiscuous CCR (15), and mannose receptors, Downloaded from which may support HIV entry through endocytosis and subsequent Cell lines and reagents escape from endosomal vesicles (16–18). The astroglioma cell lines U87MG and U251MG were obtained from the Despite the lack of clarity on how HIVenters astrocytes, our group NIH AIDS Research and Reference Reagent Program (Frederick, MD) and previously demonstrated that astrocytes support productive HIV the American Type Culture Collection (Manassas, VA), respectively. They replication if they are primed with IFN-g prior to exposure to HIV were propagated in DMEM (Life Technologies Invitrogen) supplemented with 10% heat-inactivated FBS (Sigma) and 1% penicillin-streptomycin (19). If IFN-g is provided to astrocytes post-HIV infection, it does http://www.jimmunol.org/ (Life Technologies Invitrogen). The cells were used at ∼80% confluency. not promote productive HIV replication, and the virus remains latent Human rIFN-g,GSK3b Ab (pY216), pSTAT1 (S727)-AF647 mAb, in astrocytes. Recent studies on postmortem tissue isolated from pSTAT3 (pY705)-AF488, pSTAT4 (pY693)-AF647, pSTAT5 (pTyr694)- brains of HIV+ patients with neurocognitive impairment revealed AF488, and pSTAT6 (pY641)-PE were purchased from BD Pharmingen considerable infection of astrocytes in vivo. Interestingly, the se- (San Jose, CA). pSTAT2 (pTyr690) was purchased from Cell Signaling verity of HIV-associated dementia (HAD) correlated with the de- (Danvers, MA). Allophycocyanin and FITC-conjugated goat anti-mouse Abs and FITC-bovine anti-goat IgG (H+L) Ab were purchased from gree of HIV infection of astrocytes and their close proximity to Jackson ImmunoResearch Laboratory (West Grove, PA). Fludarabine perivascular macrophages (20). These studies suggested that under (FLUD) was purchased from Sigma-Aldrich. STAT3 inhibitor V, STATtic, the appropriate environmental milieu, astrocytes can support STAT5inhibitor, and GSK3b inhibitor IX were purchased from CalBiochem/ productive HIV replication. The mechanism by which signals, EMD Biosciences (Gibbstown, NJ). hDKK1 (DKK1)-neutralizing Ab was by guest on September 24, 2021 purchased from R&D Systems (Minneapolis, MN), whereas DKK1- such as IFN-g, prime astrocytes for productive HIV replication is detection Ab (clone #569559) was purchased from Abcam (Cambridge, not clear. Astrocytes express robust levels of b-catenin signaling, MA). Dkk1 ELISA was purchased from RayBiotech (Norcross, GA) and which causes repression of HIV replication in astrocytes (21, 22) used as recommended. HIV p24 concentration was measured by conven- and PBMCs (23, 24). This finding suggests a possible interface tional ELISA from the AIDS and Cancer Virus Program (Science Appli- between the b-catenin pathway and the IFN-g–signaling path- cations International Corp., Frederick, MD). way that can impact HIV replication in astrocytes. DNA constructs and transfection The b-catenin pathway is the canonical pathway of Wnt sig- U87MG and U251MG astroglioma cell lines and primary human astrocytes naling. It is emerging as an important regulator of neurodegen- were transiently transfected using either the LT-1 DNA transfection reagent erative diseases (25–28). The b-catenin signal-transduction cascade (Mirus Bio, Madison, WI) or the Amaxa nucleofection protocol (Amaxa, is multifaceted and is described in detail elsewhere (29). Briefly, Gaithersburg, MD), as recommended by the manufacturer. To measure b- 6 the canonical pathway is initiated by the binding of Wnt pro- catenin–dependent signaling activity, 5 3 10 cells were transfected with teins (a family of 19 soluble secreted glycoproteins) to Frizzled 10 mg TOPflash reporter construct (Millipore, Billerica, MA). TOPflash construct consists of two sets of three TCF/LEF-binding sites linked to (G-coupled seven transmembrane protein receptor, Fz) and low- a luciferase reporter. The cells were also cotransfected with 1 ng Renilla density lipoprotein receptor-related protein 5 or 6 coreceptors. construct (Promega, Madison, WI) to normalize for transfection efficiency This event leads to the inhibition of a multiprotein b-catenin de- and GFP (pMaxGFP; Lonza, Biologics, Portsmouth, NH) to equalize the struction complex (glycogen synthase kinase-3b [GSK3b], axin, amount of total DNA used per transfection condition. Firefly and Renilla luciferase activity was measured using dual luciferase assay reporter sys- adenomatous polyposis coli, casein kinase 1), resulting in accu- tem (Promega). Where indicated, cells were transfected with TOPflash and mulation of a stable/hypophosphorylated b-catenin. Active (hypo- Renilla, with or without a constitutively active b-catenin construct (Cara phosphorylated) b-catenin functions as a coactivator for T cell Gottardi, Northwestern University, Chicago, IL) or a dominant-negative factor/lymphoid enhancer (TCF/LEF) transcription factors and, (DN) mutant TCF-4 construct (James O’Kelly, University of California, along with coactivators (CBP and p300), leads to target gene Los Angeles). The constitutively active b-catenin plasmid contains a ser- ine-to-tyrosine mutation at position 33 that protects the protein from transcription. b-catenin target genes impact cell differentiation, proteosomal degradation. DN TCF-4 constructs lack the N-terminal 31 aa communication, apoptosis/survival, and proliferation (30, 31). required for b-catenin binding. Active b-catenin can also bind to cadherins in the cell membrane, IFN-g treatment and HIV infection along with actin, to provide structural support for adhesion. In this study, we determined the mechanism by which IFN-g Astrocytes were pretreated with IFN-g (100 ng/ml) or left untreated for 24 promotes productive HIV replication in astrocytes. Our study dem- h prior to HIV infection. IFN-g was maintained postinfection. HIV in- fection was carried out using IFN-g–primed astrocytes at 80% confluency onstrated a link between IFN-g signaling and b-catenin signaling and incubating the cells with HIVBal (NIH AIDS Research and Reference that impacts HIV replication in astrocytes and may have a greater Reagents Program, Germantown, MD) at 10 ng HIV p24/1 3 106 cells for biologic impact on mechanisms of viral pathogenesis in the CNS. 24 h. Postinfection, the cells were washed extensively with 13 PBS and The Journal of Immunology 6773 propagated in the presence of IFN-g (100 ng/ml). At day 7 postinfection, b-catenin in human primary fetal astrocytes (PFA), thereby in- HIV p24 was monitored by conventional ELISA, according to recom- creasing restricted HIV replication in astrocytes. PFA were mendations of the manufacturer (AIDS and Cancer Virus Program, Science cotransfected with a TCF/LEF firefly luciferase construct (TOP- Applications International Corp., Frederick, MD). flash) and a control reporter (Renilla luciferase) and then treated Immunofluorescence staining and flow cytometry analysis or not with IFN-g. The TOPflash reporter is an indicator of basal To detach astrocytes without cleaving surface proteins, they were incubated and inducible levels of b-catenin–dependent signaling. At 24 h with 1 mM EDTA for 5 min and then washed and suspended in 13 PBS. post–IFN-g treatment, IFN-g markedly reduced b-catenin sig- Cells were stained with appropriate target Abs and isotype Abs using naling by ∼38% (Fig. 1A). IFN-g–mediated inhibition of b-cat- conventional surface- and/or intracellular-staining methods. When both enin signaling in PFA was also consistent with a reduction in surface and intracellular staining was desired, cells were first fixed and permeabilized using BD Cytofix/Cytoperm Fixation and Permeabilization active hypophosphorylated b-catenin, as evaluated by intracellular Solution (BD Pharmingen), followed by staining for intracellular proteins. flow cytometry (Fig. 1B). We also confirmed the ability of IFN-g Cells were then washed extensively with 13 PBS to remove excess Ab, to diminish b-catenin signaling in U251MG astroglioma cells, as stained for extracellular targets, and fixed with 2% formaldehyde. Fluo- demonstrated by ∼38% decline in TOPflash activity at 24 h rescence was evaluated with a FACSCalibur flow cytometer, and data were postexposure (Fig. 1C). Kinetics of IFN-g–mediated reduction in analyzed using FlowJo software (Tree Star, Ashland, OR). the expression of active b-catenin indicated that this process is STATistical analysis initiated as early as 1 h posttreatment, and ∼45% reduction in STATistical analyses were performed using Prism software (GraphPad Prism, active b-catenin expression is achieved by 48 h post–IFN-g ex- San Diego, CA). Untreated and treated (IFN-g with or without inhibitor) posure in U251MG cells (Fig. 1D). Specificity of endogenous groups were compared using the Student t test when the data were normally b-catenin–signaling activity in astrocytes is demonstrated by Downloaded from distributed. When the data were not normally distributed, the two groups comparing the activity of the TOPflash construct with a FOPflash were compared using the nonparametric Mann–Whitney U test. All tests were two-tailed, and a p value ,0.05 was considered significant. construct. FOPflash is a negative control for TOPflash; it consists of the same backbone vector of TOPflash linked to firefly lucif- erase but with mutated TCF/LEF-binding sites (Fig. 1E). This Results construct illustrates the expected basal/low activity of backbone IFN-g–mediated induction of HIV replication in astrocytes is vector in these cells (Fig. 1E). http://www.jimmunol.org/ b-catenin–signaling dependent To evaluate whether IFN-g–mediated induction of HIV repli- Active b-catenin signaling inhibits HIV replication in astrocytes cation in astrocytes is dependent on downregulation of b-catenin, and PBMCs (21–24). We evaluated whether IFN-g downregulates we used both gain- and loss-of-function studies. For gain-of- by guest on September 24, 2021

FIGURE 1. IFN-g downregulates b-catenin signaling pathway. PFA (A) or U251MG astroglioma cells (C) were left untreated or treated with IFN-g (100 ng/ml) for 24 h prior to transfection with TOPflash luciferase and Renilla luciferase constructs. After resting for 4 h, the cells were cultured with or without initial treatment of IFN-g. Dual luciferase activity was measured 24 h later. Data shown are normalized to Renilla activity. Background level of dual luciferase reading is between 0.35–0.13 and is indicated in first two columns of A and C.PFA(B) or U251MG cells (D) were treated with or without IFN-g for 48 h (B) or for 1, 24, or 48 h (D), and expression of hypophosphorylated/active b-catenin level was measured by conventional intracellular flow cytometry. E, PDA or U87MG cells were transfected with TOPflash or FOPflash with Renilla. Firefly luciferase activity over Renilla activity was measured 24 h posttransfection. Data represent a minimum of three experiments. *p , 0.05. 6774 INTERACTION BETWEEN IFN-g AND b-CATENIN SIGNALING IN CNS

FIGURE 2. Impact of b-catenin and TCF-4 on IFN-g–mediated induction of HIV in astrocytes. PFA (A) or U87MG astrocytoma cells (B) were transfected with GFP, a constitutively active b-catenin construct (b-catenin pcDNA), a DN construct of TCF-4, or were mock transfected. Transfected cells were treated or not with IFN-g for 24 h. Subsequently, the cells were infected with HIVBal, and HIV p24 level was measured at day 6 postinfection by conventional ELISA. Data represent a minimum of three experiments. *p , 0.05, compared with untreated samples. Downloaded from function studies, we transfected PFA (Fig. 2A) or U87MG astro- IFN-g to induce HIV replication in astrocytes is dependent on its glioma cells (Fig. 2B) with a constitutively active construct of ability to downregulate b-catenin signaling. Inhibiting b-catenin b-catenin. For loss-of-function studies, we transfected the cells signaling, through DN TCF-4 expression, had no effect on IFN-g– with a DN construct of TCF-4. Overexpressing b-catenin abro- mediated induction of HIV replication in both cell types (Fig. 2). gated the ability of IFN-g to induce HIV replication in both PFA This is likely because IFN-g inhibits b-catenin signaling (Fig. 1),

and U87MG (Fig. 2). These data demonstrated that the ability of and further inhibition of b-catenin signaling by DN TCF-4 http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 3. IFN-g induces DKK1 expression and has minimal effects on GSK3b. PFA or U251MG cells were treated with or without IFN-g, and the expression of DKK1 was measured by flow cytometry (A, C) or ELISA (B, D) at 48 h. Endogenous level of DKK1 in untreated PFA is 863 pg/ml and of U251 is 1248 pg/ml. E, Active GSK3b level was measured in U251 cells at 1 h post–IFN-g exposure, with or without the addition of a GSK3b-specific inhibitor. Data represent a minimum of three experiments. *p , 0.05, compared with untreated samples. MFI, mean fluorescence intensity. The Journal of Immunology 6775 expression did not have additional effects over that already con- IFN-g induction of HIV replication in astrocytes is dependent ferred by IFN-g treatment alone. It is interesting to note that on its ability to induce DKK1 and STAT3 inhibiting endogenous b-catenin activity enhanced HIV replica- We determined whether the ability of IFN-g to increase DKK1 and, tion in untreated cultures (Fig. 2). This observation is consistent to a lesser extent, GSK3b may play a role in the mechanism by with our previous studies demonstrating that b-catenin is an en- which IFN-g overcomes restricted HIV replication in astrocytes. dogenous factor that represses HIV replication and that its in- Primary astrocytes were pretreated with IFN-g, with or without hibition promotes HIV replication in a number of cell types, neutralizing Abs against DKK-1 (aDKK-1) or a GSK3b inhibitor including astrocytes (21, 23). (G3I). The cells were then infected with HIVBal, and HIV p24 IFN-g inhibits b-catenin signaling through induction of DKK1, level was measured 6 d postinfection. IFN-g pretreatment induced an antagonist of the b-catenin pathway HIV replication in primary astrocytes by 4-fold (Fig. 5A). Inhib- iting DKK-1 reduced the ability of IFN-g to induce HIV repli- To determine how IFN-g downregulates b-catenin–signaling ac- cation by 50% (Fig. 5A). Using G3I (Fig. 3E) had no statistically tivity, we evaluated the impact of IFN-g on two prominent significant effect on the IFN-g–mediated induction of HIV repli- antagonists of the b-catenin pathway: DKK1 and GSK3b. DKK1 cation. This observation was also consistent in U87MG cells (Fig. antagonizes b-catenin signaling by depleting frizzled coreceptors 5B). (low-density lipoprotein receptor-related protein), thus inhibiting Because inhibiting DKK-1 did not completely abrogate the frizzled activation through Wnt ligands (29). Following casein ability of IFN-g to promote HIV-productive replication in astro- kinase 1-mediated phosphorylation of b-catenin at serine 45, cytes (Fig. 5), we investigated the contribution of classical IFN-g GSK3b phosphorylates b-catenin at Thr 41, Ser 33, and Ser 37, Downloaded from signaling on its ability to enhance HIV replication in astrocytes. which tags b-catenin for ubiquitination by bTrCP and proteomic Within 30 min of exposure, IFN-g activated STAT 1 and STAT 3 degradation. IFN-g–treated PFA demonstrated a significant in- and had no effect on STAT 2, 4, 5, or 6 in PFA, U251, and U87MG duction in DKK1 expression, as measured by both flow cytometry cells (data not shown). STAT3 inhibitor (S3I) reduced IFN-g– and ELISA, and this was consistent between PFA and U251MG mediated induction of HIV replication by ∼32%, whereas inhib- cells (Fig. 3A–D). Similar results were observed in U87MG (data iting STAT1 by FLUD had no effect (Fig. 6). Further, combining

not shown). Further, addition of neutralizing Ab against DKK1 http://www.jimmunol.org/ inhibitors of STAT3 and DKK1 abrogated the effect of IFN-g on abrogated the ability of IFN-g to induce DKK1 and reduced the increased HIV replication in astrocytes (Fig. 6). These data level of DKK1 from untreated cultures (Fig. 3B,3D). These data demonstrated that the ability of IFN-g to induce productive HIV suggested that astrocytes constitutively express DKK1, which is replication in astrocytes is mediated by STAT 3 and DKK1. consistent with the knowledge that DKK1 is a target gene of the Given that IFN-g induction of DKK1 is a prominent pathway b-catenin pathway and regulates the expression of this pathway in by which it downregulates b-catenin signaling and subsequently a feedback-loop mechanism (29). Active GSK3b expression was enhances HIV replication in astrocytes, we evaluated whether modestly increased by IFN-g at 1 h posttreatment, but this in- IFN-g induction of DKK1 and inhibition of b-catenin are STAT 3 duction was transient and returned to background level by 2 h dependent. Inhibition of STAT3 abrogated the ability of IFN-g to (Fig. 3E). by guest on September 24, 2021 downregulate b-catenin (Fig. 7A) and induce DKK-1 (Fig. 7B). To determine whether IFN-g inhibition of b-catenin signaling is STAT1 had no effect on IFN-g induction of DKK1 and inhibition mediated by its induction of DKK1, PFA were treated with IFN-g of b-catenin (data not shown). These data demonstrated that IFN- in the presence or absence of a DKK1-specific inhibitor. The ability of IFN-g to inhibit the b-catenin pathway was abrogated in the presence of the DKK1 inhibitor, as measured by TOPflash activity (Fig. 4). The DKK-1 inhibitor alone, in the absence of IFN-g treatment, enhanced TOPflash activity, suggesting that as- trocytes express endogenous DKK1 to regulate b-catenin–medi- ated signaling. Similar results were observed in U251MG cells (data not shown).

FIGURE 4. IFN-g inhibition of b-catenin signaling is through its in- duction of DKK-1. PFA were left untreated or were treated with IFN-g, FIGURE 5. IFN-g induces HIV replication in astrocytes via upregula- anti–DKK-1, or both IFN-g and anti–DKK-1 for 24 h prior to transfection tion of DKK1. PFA (A) or U87MG cells (B) were pretreated or not with with TOPflash and Renilla constructs. Posttransfection, the cells were IFN-g in the presence or absence of anti–DKK-1 or G3I for 24 h, infected maintained in their initial treatment for 24 h. Dual luciferase activity was with HIVBal, and cultured with initial treatment. At day 6 postinfection, measured, and TOPflash luciferase was normalized to Renilla firefly ac- HIV p24 was measured by ELISA. Data represent a minimum of three tivity. Data represent a minimum of three independent experiments. *p , independent experiments. *p , 0.05, compared with IFN-g–treated cul- 0.05, compared with untreated samples. tures alone. 6776 INTERACTION BETWEEN IFN-g AND b-CATENIN SIGNALING IN CNS

cytes are productively infected in vivo and require biologic signals to promote productive HIV replication, which may be lacking in an in vitro model system. The nature of the biologic signals promoting HIV permissiveness in astrocytes is not completely clear. We demonstrated that IFN-g may be such a signal that primes HIV productive infection in vitro (19). IFN-g levels are elevated in neuroAIDS and may drive higher levels of HIV rep- lication in astrocytes in vivo (5). Further, IFN-g is secreted by activated macrophages/microglia, which may explain the recent findings of higher levels of HIV infection in astrocytes that are in close proximity to macrophages/microglia (20). Astrocytes themselves secrete IFN-g, which may function in an autocrine fashion to enhance HIV infection in these cells. Astrocytes have robust b-catenin signaling (21), which is in- FIGURE 6. IFN-g enhancement of HIV replication in astrocytes is versely correlated with HIV replication in a number of cell types, DKK1 and STAT 3 dependent. PFA were treated or not with IFN-g in the presence of S3I, STAT1 inhibitor (FLUD), or S3I, FLUD and anti–DKK-1 including astrocytes (21, 23). Specifically, inhibiting b-catenin signaling in astrocytes through the use of a DN construct of for 48 h prior to HIVBal infection. At day 6 postinfection, HIV p24 was measured by ELISA. Data represent a minimum of three independent b-catenin or TCF-4 promoted HIV productive replication in experiments. *p , 0.05, compared with IFN-g–treated cultures alone. astrocytes. Because IFN-g inhibits b-catenin, which is a negative Downloaded from regulator of HIV replication, we evaluated whether IFN-g pro- g–mediated inhibition of b-catenin and induction of DKK-1 are motes HIV replication in astrocytes by inhibiting b-catenin and also STAT3 dependent. Collectively, these findings demonstrated determined the mechanism by which it does so. In this study, we an interaction between two prominent signaling pathways, b- demonstrated that the ability of IFN-g to mediate productive HIV catenin and IFN-g signaling, that interface with each other to replication in astrocytes occurs through inhibition of the b-cat- impact the outcome of HIV in the CNS. enin–signaling pathway in a STAT3-dependent manner. Further, http://www.jimmunol.org/ IFN-g–mediated STAT3 activation induces an antagonist of the Discussion b-catenin pathway, DKK-1. Both IFN-g induction of STAT3 and Using sophisticated assessment of HIV infection of postmortem DKK-1 are critical in its ability to promote HIV replication in tissue, Churchill et al. (20) recently demonstrated that #19% of astrocytes. This finding is especially intriguing because it points to GFAP+ astrocytes are infected by HIV. The level of HIV infection interplay between b-catenin and IFN-g signaling leading to en- of astrocytes was highest among those in close proximity to hanced HIV replication. Our data also add to the body of evidence macrophages/microglia. Although a disconnect existed between pointing to STAT1-independent mechanisms of IFN-g signaling in vitro and in vivo data with regard to whether astrocytes are events that lead to IFN-g–dependent effects and infected by HIV, these postmortem data demonstrated that astro- (6). by guest on September 24, 2021

FIGURE 7. IFN-g activation of STAT3 in- duces DKK1 expression and inhibits b-catenin. U87MG cells and PFA were treated or not with IFN-g in the presence of S3I for 48 h. Sub- sequently, active b-catenin level was measured by flow cytometry (A), and DKK1 level was measured by ELISA (B). Data represent a min- imum of three independent experiments. *p , 0.05, compared with IFN-g–treated cultures alone. The Journal of Immunology 6777

IFN-g inhibition of b-catenin signaling demonstrates a signifi- signaling in astrocytes to promote productive HIV replication and cant cross-talk between the IFN-g and b-catenin pathways. Al- negative effects that may ensue as a result of this downregulation. though, classically, the presence or absence of Wnt ligands Wnt signaling regulates hippocampal neurogenesis in the adult dictates whether the b-catenin pathway is engaged, there is a brain (42) and is a well-established prosurvival pathway. These growing appreciation for Wnt ligand-independent regulation of findings suggest that signals that downregulate b-catenin signal- b-catenin. Extensive cross-talk exists between the b-catenin ing, including IFN-g, may have negative effects on neurogenesis pathway and other signal-transduction cascades, including the and cell survival while enhancing HIV replication. Conversely, PI3K/Akt and p38 MAPK pathways, which interact with the ca- b-catenin signaling may be manipulated to favor neurogenesis and nonical pathway by converging on a third signaling partner: neuroprotection against toxic insults in a number of neurodegen- GSK3b. This study adds to the growing number of pathways that erative diseases, including HAD and/or HAND. can regulate b-catenin signaling, independent of Wnt ligand en- The link that we demonstrated in this study between IFN-g gagements. Further, b-catenin is tightly regulated to avoid ab- and b-catenin–signaling pathway suggests that IFN-g may have errant activation. A number of proteins in the b-catenin–signaling broader roles than was previously appreciated. Understanding the pathway are regulated at the gene level by b-catenin/TCF tran- interactions between IFN-g and b-catenin signaling will have scriptional regulation via the presence of LEF/TCF-binding sites a broader impact on viral infection, as well as on understanding in these genes. DKK1 is one of these genes and is positively the normal biology of immune or brain cells at the interface of regulated by b-catenin/TCF-mediated gene regulation. Because IFN-g and b-catenin–dependent pathways. DKK1 is an antagonist of the b-catenin pathway, this may be a mechanism by which b-catenin is self-regulated to avoid over- Acknowledgments Downloaded from activation that can lead to uncontrolled cell survival/proliferation We thank Dr. Cara Gottardi (Northwestern University, Chicago, IL) for pro- and, ultimately, oncogenesis. Although IFN-g inhibited active viding a constitutively active b-catenin construct and Dr. James O’Kelly b-catenin expression and signaling, it induced DKK1 protein. This (University of California, Los Angeles) for providing a DN-mutant con- finding suggested that, in addition to b-catenin/TCF gene regu- struct of TCF-4. lation of DKK1, IFN-g may be engaging another pathway by which it leads to induction of DKK1, whether via an alternative Disclosures http://www.jimmunol.org/ mechanism of DKK1 gene regulation or posttranscriptional reg- The authors have no financial conflicts of interest. ulatory events. Greater understanding of the interplay between b-catenin and other signaling pathways, including the IFN-g pathway, may provide tools for enhancing function and survival of References 1. Boehm, U., T. Klamp, M. Groot, and J. C. Howard. 1997. Cellular responses to neurons and glia, as well as manipulating HIV replication in the interferon-gamma. Annu. Rev. Immunol. 15: 749–795. CNS reservoir. 2. Lau, L. T., and A. C. Yu. 2001. Astrocytes produce and release interleukin-1, Astrocytes make up 40–60% of brain cells and play vital interleukin-6, tumor necrosis factor alpha and interferon-gamma following traumatic and metabolic injury. J. Neurotrauma 18: 351–359. functions in maintaining brain homeostasis, which suggests that 3. Ullian, E. M., S. K. Sapperstein, K. S. Christopherson, and B. A. Barres. 2001. any level of HIV replication from astrocytes in vivo may have Control of synapse number by glia. Science 291: 657–661. by guest on September 24, 2021 dramatic consequences in the brain. Indeed, dysregulation of 4. Lane, T. E., M. J. Buchmeier, D. D. Watry, and H. S. Fox. 1996. Expression of inflammatory cytokines and inducible nitric oxide synthase in brains of SIV- astrocytes is associated with a number of neurodegenerative dis- infected rhesus monkeys: applications to HIV-induced central nervous system eases, including HAD. In HAD, astrogliosis, characterized by disease. Mol. Med. 2: 27–37. hypertrophy, increased GFAP immunoreactivity, enhanced pro- 5. Shapshak, P., R. Duncan, A. Minagar, P. Rodriguez de la Vega, R. V. Stewart, and K. Goodkin. 2004. Elevated expression of IFN-gamma in the HIV-1 infected liferation, and apoptosis, is one of the hallmarks of HIV infiltration brain. Front. Biosci. 9: 1073–1081. in the brain and the severity of encephalopathy (33). However, 6. Ramana, C. V., M. P. Gil, R. D. Schreiber, and G. R. Stark. 2002. STAT1- the exact mechanism by which astrocytes contribute to HIV neu- dependent and -independent pathways in IFN-gamma-dependent signaling. Trends Immunol. 23: 96–101. ropathogenesis is not clear. Astrocytes may contribute to the re- 7. Choi, J. S., S. Y. Kim, J. H. Cha, Y. S. Choi, K. W. Sung, S. T. Oh, O. N. Kim, lease of neurotoxins (gp120, Tat) and inflammatory cytokines/ J. W. Chung, M. H. Chun, S. B. Lee, and M. Y. Lee. 2003. Upregulation of gp130 chemokines, such as TNF-a, IFN-g, and MCP-1, that cause dys- and STAT3 activation in the rat hippocampus following transient forebrain is- chemia. Glia 41: 237–246. regulation of neurons. Astrocytes themselves may be dysregulated 8. Su, Z., Y. Yuan, L. Cao, Y. Zhu, L. Gao, Y. Qiu, and C. He. 2010. Triptolide by HIV [e.g., their inability to scavenge for glutamate post-HIV promotes spinal cord repair by inhibiting astrogliosis and inflammation. Glia 58: infection and/or exposure (34)]. We propose that IFN-g may also 901–915. 9. Bromberg, J. F., M. H. Wrzeszczynska, G. Devgan, Y. Zhao, R. G. Pestell, contribute to the dysregulation of astrocytes in the context of HIV C. Albanese, and J. E. Darnell Jr. 1999. STAT3 as an oncogene. Cell 98: 295– by downregulating an important prosurvival-signaling pathway. 303. Wnt/b-catenin plays a key role in axonal remodeling and regu- 10. Heaton, R. R., D. Franklin, D. Clifford, S. Woods, M. Rivera Mind, O. Vigil, M. Taylor, T. Marcotte, H. Atkinson, and I. Grant. 2009. HIV-associated Neu- lation of synaptic connectivity in the CNS (35). Activation of Wnt rocognitive Impairment Remains Prevalent in the Era of Combination ART: The signaling by exogenous molecules, such as LiCl or Wnt-3a, pro- CHARTER Study. 16th conference on retroviruses and opportunistic infections tects cells from a number of toxic insults, such as glutamate, N- abstract 154. 11. Geleziunas, R., H. M. Schipper, and M. A. Wainberg. 1992. Pathogenesis and methyl-D-aspartate, calcium, and b-amyloid and deprivation of therapy of HIV-1 infection of the central nervous system. AIDS 6: 1411–1426. KCl, serum, and nerve growth factor (36, 37). In a mouse model 12. Markiewicz, I., and B. Lukomska. 2006. The role of astrocytes in the physiology of neuroAIDS, LiCl was able to restore loss of microtubule- and pathology of the central nervous system. Acta Neurobiol. Exp. (Warsz.) 66: + 343–358. associated protein-2 neurites and synaptic density that is often 13. Becher, B., A. Prat, and J. P. Antel. 2000. Brain-immune connection: immuno- observed with HIV invasion of the CNS (38). Lithium remarkably regulatory properties of CNS-resident cells. Glia 29: 293–304. improved neurocognition in HIV+ patients in a small clinical trial 14. Aloisi, F. 1999. The role of microglia and astrocytes in CNS immune surveil- lance and immunopathology. Adv. Exp. Med. Biol. 468: 123–133. (39). Our previous studies defined an antiviral effect of LiCl that 15. Neil, S. J., M. M. Aasa-Chapman, P. R. Clapham, R. J. Nibbs, A. McKnight, and is b-catenin pathway dependent (39–41). Many of the neuro- R. A. Weiss. 2005. The promiscuous CC chemokine receptor D6 is a functional protective effects of lithium in the context of neuroAIDS seem to coreceptor for primary isolates of human immunodeficiency virus type 1 (HIV-1) and HIV-2 on astrocytes. J. Virol. 79: 9618–9624. be driven by its effects on b-catenin (38). As such, there seems to 16. Liu, Y., H. Liu, B. O. Kim, V. H. Gattone, J. Li, A. Nath, J. Blum, and J. J. He. be an intricate balance between downregulation of b-catenin 2004. CD4-independent infection of astrocytes by human immunodeficiency 6778 INTERACTION BETWEEN IFN-g AND b-CATENIN SIGNALING IN CNS

virus type 1: requirement for the human mannose receptor. J. Virol. 78: 4120– 29. Chien, A. J., W. H. Conrad, and R. T. Moon. 2009. A Wnt survival guide: from 4133. flies to human disease. J. Invest. Dermatol. 129: 1614–1627. 17. Vijaykumar, T. S., A. Nath, and A. Chauhan. 2008. Chloroquine mediated mo- 30. Moon, R. T., J. D. Brown, and M. Torres. 1997. WNTs modulate cell fate and lecular tuning of astrocytes for enhanced permissiveness to HIV infection. Vi- behavior during vertebrate development. Trends Genet. 13: 157–162. rology 381: 1–5. 31. Miller, J. R., and R. T. Moon. 1996. Signal transduction through beta-catenin and 18. Permanyer, M., E. Ballana, and J. A. Este´. 2010. Endocytosis of HIV: anything specification of cell fate during embryogenesis. Genes Dev. 10: 2527–2539. goes. Trends Microbiol. 18: 543–551. 32. Lamba, S., V. Ravichandran, and E. O. Major. 2009. Glial cell type-specific 19. Carroll-Anzinger, D., and L. Al-Harthi. 2006. Gamma interferon primes pro- subcellular localization of 14-3-3 zeta: an implication for JCV tropism. Glia ductive human immunodeficiency virus infection in astrocytes. J. Virol. 80: 541– 57: 971–977. 544. 33. Minagar, A., P. Shapshak, R. Fujimura, R. Ownby, M. Heyes, and C. Eisdorfer. 20. Churchill, M. J., S. L. Wesselingh, D. Cowley, C. A. Pardo, J. C. McArthur, 2002. The role of macrophage/microglia and astrocytes in the pathogenesis of B. J. Brew, and P. R. Gorry. 2009. Extensive astrocyte infection is prominent in three neurologic disorders: HIV-associated dementia, Alzheimer disease, and human immunodeficiency virus-associated dementia. Ann. Neurol. 66: 253–258. multiple sclerosis. J. Neurol. Sci. 202: 13–23. 21. Carroll-Anzinger, D., A. Kumar, V. Adarichev, F. Kashanchi, and L. Al-Harthi. 34. Zhou, B. Y., Y. Liu, B. Kim, Y. Xiao, and J. J. He. 2004. Astrocyte activation and 2007. Human immunodeficiency virus-restricted replication in astrocytes and the dysfunction and neuron death by HIV-1 Tat expression in astrocytes. Mol. Cell. ability of gamma interferon to modulate this restriction are regulated by Neurosci. 27: 296–305. a downstream effector of the . J. Virol. 81: 5864–5871. 35. Dewhurst, S., S. B. Maggirwar, G. Schifitto, H. E. Gendelman, and 22. Wortman, B., N. Darbinian, B. E. Sawaya, K. Khalili, and S. Amini. 2002. H. A. Gelbard. 2007. Glycogen synthase kinase 3 beta (GSK-3 beta) as a ther- apeutic target in neuroAIDS. J. Neuroimmune Pharmacol. 2: 93–96. Evidence for regulation of long terminal repeat transcription by Wnt transcrip- 36. Gould, T. D., and H. K. Manji. 2002. The Wnt signaling pathway in bipolar tion factor TCF-4 in human astrocytic cells. J. Virol. 76: 11159–11165. disorder. Neuroscientist 8: 497–511. 23. Kumar, A., A. Zloza, R. T. Moon, J. Watts, A. R. Tenorio, and L. Al-Harthi. 37. Inestrosa, N. C., S. Urra, and M. Colombres. 2004. Acetylcholinesterase 2008. Active beta-catenin signaling is an inhibitory pathway for human immu- (AChE)–amyloid-beta-peptide complexes in Alzheimer’s disease. the Wnt sig- nodeficiency virus replication in peripheral blood mononuclear cells. J. Virol. 82: naling pathway. Curr. Alzheimer Res. 1: 249–254. 2813–2820. 38. Dou, H., B. Ellison, J. Bradley, A. Kasiyanov, L. Y. Poluektova, H. Xiong, 24. Salim, A., and L. Ratner. 2008. Modulation of beta-catenin and E-cadherin in- S. Maggirwar, S. Dewhurst, H. A. Gelbard, and H. E. Gendelman. 2005. Neu- Downloaded from teraction by Vpu increases human immunodeficiency virus type 1 particle re- roprotective mechanisms of lithium in murine human immunodeficiency virus-1 lease. J. Virol. 82: 3932–3938. encephalitis. J. Neurosci. 25: 8375–8385. 25. Inestrosa, N. C., L. Varela-Nallar, C. P. Grabowski, and M. Colombres. 2007. 39. Gallicchio, V. S., M. L. Cibull, N. K. Hughes, and K. F. Tse. 1993. Effect of Synaptotoxicity in Alzheimer’s disease: the Wnt signaling pathway as a molec- lithium in murine immunodeficiency virus infected animals. Pathobiology 61: ular target. IUBMB Life 59: 316–321. 216–221. 26. De Ferrari, G. V., and R. T. Moon. 2006. The ups and downs of Wnt signaling in 40. Everall, I. P., C. Bell, M. Mallory, D. Langford, A. Adame, E. Rockestein, and prevalent neurological disorders. Oncogene 25: 7545–7553. E. Masliah. 2002. Lithium ameliorates HIV-gp120-mediated neurotoxicity. Mol. 27. Caricasole, A., A. Bakker, A. Copani, F. Nicoletti, G. Gaviraghi, and

Cell. Neurosci. 21: 493–501. http://www.jimmunol.org/ G. C. Terstappen. 2005. Two sides of the same coin: Wnt signaling in neuro- 41. Maggirwar, S. B., N. Tong, S. Ramirez, H. A. Gelbard, and S. Dewhurst. 1999. degeneration and neuro-oncology. Biosci. Rep. 25: 309–327. HIV-1 Tat-mediated activation of glycogen synthase kinase-3beta contributes to 28. Caricasole, A., A. Copani, A. Caruso, F. Caraci, L. Iacovelli, M. A. Sortino, Tat-mediated neurotoxicity. J. Neurochem. 73: 578–586. G. C. Terstappen, and F. Nicoletti. 2003. The Wnt pathway, cell-cycle activation 42. Lie, D. C., S. A. Colamarino, H. J. Song, L. De´sire´, H. Mira, A. Consiglio, and beta-amyloid: novel therapeutic strategies in Alzheimer’s disease? Trends E. S. Lein, S. Jessberger, H. Lansford, A. R. Dearie, and F. H. Gage. 2005. Wnt Pharmacol. Sci. 24: 233–238. signalling regulates adult hippocampal neurogenesis. Nature 437: 1370–1375. by guest on September 24, 2021