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Canonical Type I IFN Signaling in Simian Immunodeficiency Virus-Infected Macrophages Is Disrupted by Astrocyte-Secreted CCL2 This information is current as of September 27, 2021. Luna Alammar Zaritsky, Lucio Gama and Janice E. Clements J Immunol published online 9 March 2012 http://www.jimmunol.org/content/early/2012/03/09/jimmun ol.1103024 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 9, 2012, doi:10.4049/jimmunol.1103024 The Journal of Immunology

Canonical Type I IFN Signaling in Simian Immunodeﬁciency Virus-Infected Macrophages Is Disrupted by Astrocyte-Secreted CCL2

Luna Alammar Zaritsky,* Lucio Gama,* and Janice E. Clements*,†

HIV-associated neurologic disorders are a mounting problem despite the advent of highly active antiretroviral therapy. To address mechanisms of HIV-associated neurologic disorders, we used an SIV pigtailed macaque model to study innate immune responses in brain that suppress viral replication during acute infection. We previously reported that during acute infection in brain, noncanonical type I IFN signaling occurs, where IFN-b mRNA is induced while IFN-a is simultaneously suppressed. Two downstream IFN-stimulated genes, MxA and TRAIL, also show differential expression patterns. In this study, we show that differential signaling is due to interactions between macrophages and astrocytes. Astrocytes produce high levels of CCL2 upon SIV infection, Downloaded from which binds to CCR2 receptors on macrophages, leading to a selective suppression of IFN-a and the IFN-stimulated gene TRAIL while simultaneously inducing IFN-b and MxA. The interactions between chemokine and cytokine pathways are a novel ﬁnding that may speciﬁcally occur in the CNS. The Journal of Immunology, 2012, 188: 000–000.

ype I IFNs are the ﬁrst line of defense against viral IFN receptor exist, which enables IFN-b to upregulate a subset of

infections, including HIV and SIV. Canonical type I IFN antiviral genes, such as MxA. Other groups have also found that http://www.jimmunol.org/ T signaling begins with the production of IFN-b upon despite the suppression of the JAK/STAT pathway, IFN-b still stimulation of pattern recognition receptors (PRRs). IFN-b is then upregulates a subset of antiviral ISGs through alternative path- secreted and binds the IFN receptor in an autocrine and paracrine ways, such as MAPK and PI3K (3). These results reveal that manner where it activates the JAK/STAT pathway, leading to the highly regulated mechanisms controlling IFN signaling occur expression of IFN-a and a plethora of antiviral IFN-stimulated during acute SIV infection in the brain, where the JAK/STAT genes (ISGs). IFN-a is then secreted and binds to the IFN re- pathway leading to IFN-a and TRAIL is suppressed, whereas ceptor where it perpetuates the IFN signaling cascade in a positive other IFN-b–stimulated pathways downstream of the IFN receptor feedback loop (1). are induced. Our group has made the novel discovery that type I IFN signaling Why this differential regulation occurs in the CNS and not in by guest on September 27, 2021 in the CNS in response to SIV infection occurs in a more complex, peripheral tissues, such as the lung, is most likely a consequence of noncanonical manner (2). Using an accelerated, consistent pig- the effects IFNs and their ISGs have in the brain. Whereas IFN-b tailed macaque SIV model of HIV-associated neurologic disease, has been associated with neuroprotection and the stimulation of we have shown that whereas IFN-b is induced during acute SIV anti-inflammatory mediators, IFN-a and Tyk2 have been associ- infection in brain, IFN-a is simultaneously downregulated and ated with neurotoxicity, inflammation, cell death, and neuro- associated with a suppression of signaling modulators downstream inflammatory diseases such as HIV dementia and multiple of the IFN receptor, including Tyk2, STAT1, and IRF7. This sclerosis (4–10). TRAIL is a gene associated with cell death as results in differential regulation of ISGs as well, with MxA being well. Therefore, this differential pattern of IFN signaling in brain induced along with IFN-b, whereas TRAIL is suppressed along may be an evolutionary mechanism to induce an antiviral response with IFN-a. These results indicate that suppression of JAK/STAT mediated by IFN-b without inducing the inflammatory and de- signaling leading to IFN-a and TRAIL does not result in complete structive effects mediated by IFN-a, TRAIL, and the JAK/STAT shutdown of antiviral signaling, as IFN-b and MxA are still in- pathway. duced. Rather, accessory signaling pathways downstream of the To investigate the mechanism behind this differential IFN signaling, we examined the two cell types involved in HIV and SIV infection in brain: macrophages and astrocytes. HIV and SIV in- *Department of Molecular and Comparative Pathobiology, The Johns Hopkins School of Medicine, Baltimore, MD 21205; and †Department of Neurology and fection of the CNS occurs during early stages of disease, as in- Pathology, The Johns Hopkins School of Medicine, Baltimore, MD 21205 dicated by production of viral mRNA and protein both in brain Received for publication October 20, 2011. Accepted for publication February 13, tissue and cerebrospinal fluid (11–13). This occurs via a “Trojan 2012. horse” mechanism, where infection of certain subpopulations of This work was supported by grants from the National Institutes of Health (NS047984, peripheral monocytes results in immune activation and upreg- NS055648, and MH070306 to J.E.C.). ulation of adhesion molecules such as VCAM-1, facilitating Address correspondence and reprint requests to Dr. Janice E. Clements, The Johns transmigration across the blood–brain barrier (14–16). These Hopkins School of Medicine, 733 N. Broadway, BRB Suite 820, Baltimore, MD 21205. E-mail address: [email protected] monocytes accumulate in the perivascular regions and undergo Abbreviations used in this article: ISG, IFN-stimulated gene; MDM, monocyte- differentiation into macrophages, which is where early HIV and derived macrophage; MOI, multiplicity of infection; p.i., postinfection; polyI:C, SIV replication takes place (17–20). Although induction of cyto- polyinosinic:polycytidylic acid; PRR, pattern recognition receptor; qRT-PCR, quan- kines such as IFN-b in macrophages has been shown to be pro- titative RT-PCR. tective against SIV infection in the brain (21, 22), uncontrolled Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 viral replication can lead to the production of proinflammatory

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103024 2 CCL2 DISRUPTS TYPE I IFN SIGNALING IN MACROPHAGES cytokines, excitatory amino acids, and molecules mediating oxi- Astrocytes. Primary rhesus macaque astrocytes (Cambrex, Walkersville, dative stress, all of which ultimately lead to neuronal death (23, MD) were cultured as previously described (39). Cells were infected with 24). Astrocytes are the most numerous cells in the brain and have SIV/17E-Fr at an MOI of 0.05 for 6 h and then washed three times with sterile PBS. Cells were then cultured in D10 (DMEM, 10% FBS, 10 mM been reported to express relatively high levels of TLR3 and the HEPES, 2 mM L-glutamine, 0.5 mg/ml gentamicin, and 2 mM Na pyru- downstream signaling adapter molecules, indicating that astrocytes, vate). Half of the media was replaced with fresh D10 every 5 d for 30 d. as well as macrophages, play a major role in the immunological Infected and uninfected cells were harvested on days 5–7, 10, 14, 21, and response to infection (25–27). HIV/SIV infection in astrocytes has 25. RNA was isolated using the RNeasy kit and treated with DNase as described earlier. Protein lysates were made as indicated earlier. For been reported in brain tissue of humans and macaques, as well as polyinosinic:polycytidylic acid (polyI:C)-treated and IFN-b–treated as- in vitro (13, 28). Upon infection, astrocytes are the major producers trocytes, cells were treated with either 10 mg/ml polyI:C or 100 U/ml IFN- of MCP-1, or CCL2, which is a chemokine that recruits monocytes b. Cells were harvested at 24, 48, and 72 h posttreatment, and RNA was expressing CCR2 into the CNS (14). Certain populations of isolated as described earlier. macrophages/microglia in brain have been shown to express CCR2 Supernatant exchange reactions. Primary MDMs were infected as indicated during SIV/HIV infection (29). Importantly, CCL2 has also been earlier. After washing off virus, cells were cultured with either normal macrophage media, supernatants from uninfected astrocytes, or super- reported to protect neurons from apoptosis as well as inhibit glu- natants from infected astrocytes. Astrocytes supernatants, including time- tamate production in response to HIV infection, making it an im- point–specific uninfected controls, were collected at 14–21 d p.i. when portant neuroinflammatory modulator (30). cells were harvested. Ultracentrifugation was done at 40,000 rpm for 2 h at Perhaps the most significant immunological role astrocytes play 4˚C on both sets of supernatants using the Sorvall Discovery 100SE (rotor TH641) (Thermo-Fischer Scientific) to pellet virus and cellular debris. in the CNS is their immunomodulatory effects on macrophages and Supernatants were then passed through an 0.22-mm filter unit, and a p27 microglia. Microglia, the resident macrophages of the brain, have analysis by ELISA (Zeptometrix) was done to verify no infectious virus Downloaded from been shown to exhibit increased inflammatory activity in vitro was present. Infected and uninfected cells were harvested 48 h p.i., and than that observed in pathologic conditions in the brain (31, 32), RNA was isolated as described earlier. largely because microglia are usually cultured alone, which does Quantitative RT-PCR not accurately portray the CNS microenvironment. However, by coculturing astrocytes with macrophages or by exchanging super- All primer and probe sequences as well as quantitative RT-PCR (qRT-PCR) protocols are described elsewhere (2). All genes were normalized to 18S natants, it was found that astrocytes release soluble factors that rRNA. SIV-infected samples were expressed as a fold induction over http://www.jimmunol.org/ attenuate the inflammatory response in macrophages. These ef- each sample’s respective uninfected control. In the case where uninfected fects include reducing the expression of inducible NO synthase samples were given a treatment, all treated samples were expressed as a and IL-12 (33, 34), increasing expression of antioxidant enzymes fold induction over untreated controls. (35), and inhibiting IFN-g–induced microglial activation (36). Western blots and Abs Also, HIV-infected monocytes that interact with astrocytes have All Western blot protocols and Abs used are described elsewhere (2). been reported to secrete decreased levels of TNF-a and eicosa- Quantitations were performed using ImageQuantTL 7.0 software on noids upon LPS stimulation (37). scanned images. Because astrocytes have been shown to attenuate the inflam-

CCL2 ELISA by guest on September 27, 2021 matory response in macrophages, we examined if these cells could selectively down modulate IFN-a, as this cytokine is a potent CCL2 was measured in the supernatants of SIV-infected and UI macro- neuroinflammatory mediator. We found that the differential IFN phages (48 h p.i.), and of SIV-infected and UI astrocytes (14 d p.i.) using response observed in vivo is attributed to astrocytic production of the Quantikine Human CCL2 ELISA (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. CCL2. When SIV-infected macrophages are exposed to CCL2, there is specific suppression of IFN-a and TRAIL while upregu- CCL2 neutralization lation of IFN-b and MxA is maintained. This not only illustrates Infected astrocyte supernatants were incubated with 50 mg/ml of anti- the novel role CCL2 plays in selectively suppressing IFN-a ex- human CCL2 neutralizing Ab (R&D Systems) or nonspecific goat IgG pression in SIV-infected macrophages but also suggests that the for 1 h at room temperature. The supernatants were then added to unin- immunomodulatory effects that astrocytes exert on macrophages fected and SIV-infected MDMs after virus had been washed off. Cells were harvested 48 h p.i., and RNA was isolated using the RNeasy kit (Qiagen) as are a key factor in maintaining an anti-inflammatory environment described earlier. during acute viral infection in the brain. Chemokine and growth factor treatment Materials and Methods Uninfected and SIV-infected MDMs were untreated or treated with either Cell culture and infections 100 ng/ml recombinant human CCL2 (ProSpec, East Brunswick, NJ), 0.2 ng/ml recombinant human TGF-b (R&D Systems), or 10 ng/ml Macrophages. Primary pigtailed macaque monocyte-derived macrophages recombinant human CCL3 (ProSpec). Cells were harvested 48 h p.i., (MDMs) were prepared from whole blood as previously described (38). On and RNA was isolated using the RNeasy kit (Qiagen) as described earlier. day 7, cells were washed three times with sterile PBS to remove non- adherent cells. On day 8, cells were infected with SIV/17E-Fr, which was CCR2 inhibitor: propagermanium grown using macaque producer cells, using an MOI of 0.05 for 6 h and then washed five times with PBS. Cells were then cultured with normal Bis[2-carboxyethylgermanium(IV) sesquioxide] was purchased from macrophage media, which consists of DMEM (Invitrogen, Grand Island, Sigma Aldrich (St. Louis, MO) and made into a 10 mg/ml stock solution in water. (Other commonly used names: propagermanium and 2-carboxy- NY), 10% FBS, 10 mM HEPES (Invitrogen), 2 mM L-glutamine (Invi- m trogen), and 0.5 mg/ml gentamicin (Invitrogen), unless otherwise indi- ethylgermasesquioxane.) A final concentration of 100 M was used to treat cated. SIV-infected and time-point–specific uninfected controls were infected and uninfected MDMs. For samples that were treated with both harvested at 24, 48, and 72 h postinfection (p.i.). RNA was isolated using propagermanium and CCL2, cells were incubated with propagermanium the RNeasy kit (Qiagen, Valencia CA) and then treated with Turbo DNase 45 min before adding CCL2. (Ambion) according to the manufacturer’s instructions. Protein lysates Statistical analysis were made using RIPA buffer (0.25% Na deoxycholate, 0.1% SDS, 25 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% Nonidet P-40 in water) For mRNA comparisons, either one-way ANOVA with Bonferroni posttest and spiked with 1 mM DTT, 0.1 mM PMSF, protease inhibitor mixture for multiple comparisons or two-way ANOVA with Bonferroni posttest (Calbiochem, Darmstadt, Germany), and phosphatase inhibitor cocktails for multiple comparisons was performed depending on the experimental (Calbiochem) just before use. conditions. For protein comparisons that only consisted of two groups The Journal of Immunology 3

(uninfected versus infected), an unpaired t test was performed. Exact dicate that both IFN-a and IFN-b are upregulated in SIV-infected statistical analysis performed for each graph is outlined in the figure leg- MDMs in vitro, and downstream IFN signaling is activated. ends. Results were considered significant for p , 0.05. IFN signaling is downregulated in SIV-infected astrocytes Results Primary rhesus astrocytes were infected with SIV (MOI 0.05) and Type I IFN signaling is upregulated in SIV-infected MDMs harvested at 2, 7, 10, 14, and 21 d p.i. for mRNA analysis. As we and other groups have shown (39, 40), whereas virus infection Because macrophages are the major target cells responsible for SIV in macrophages takes place during the course of days, infection in replication in brain, we examined SIV infection of pigtailed ma- astrocytes takes longer because restrictive factors specific to this caque macrophages in vitro. Macaque MDMs were infected with cell type severely slows down viral replication in vitro (Figs. 1A, SIV (MOI 0.05) and harvested at 24, 48, and 72 h p.i. Levels of Gag 2A). Unlike macrophages, which displayed an activated IFN re- p27 were measured in cell supernatants to quantify viral production sponse, no significant induction of IFN-a, IFN-b, or MxA mRNA (Fig. 1A), and qRT-PCR was performed for IFN-a and IFN-b.We was observed for any of the time points (Fig. 2A–D), nor was there found that at 48 h p.i., both IFN-b and IFN-a mRNA are signif- an increase in STAT1 protein or phosphorylation status in infected , , icantly upregulated 5-fold (p 0.01) and 2-fold (p 0.01), cells (Fig. 2E). TRAIL mRNA was undetectable. To determine why respectively (Fig. 1B, 1C), coinciding with the start of viral this inhibition of IFN was occurring, we measured IRF7 mRNA detection in the supernatant. mRNA levels of two ISGs, TRAIL and protein, as this is an important transcription factor in IFN-a and MxA, were also quantitated. We show that at 48 h p.i., both and IFN-b production that is both transcriptionally and transla- genes are significantly upregulated (p , 0.05, p , 0.001, re-

tionally induced upon viral infection (41). IRF7 mRNA was un- Downloaded from spectively) (Fig. 1D, 1E), contrary to our in vivo observations detectable in infected and uninfected cells, and there was no where MxA mRNA is upregulated in the same pattern as IFNB, detectable change in protein at 14 d p.i. (Fig. 2H). To ensure that whereas TRAIL, like IFN-a, is not induced (2). Because the 48-h the astrocytes were immunologically functional, uninfected cells time point exhibited the least amount of variability in gene ex- were treated with polyI:C to test pathogen sensing activity and with pression between replicates, this time point was chosen for sub- IFN-b to test stimulation through the IFN receptor. Both treatments sequent experiments. By 72 h p.i., cell death started occurring in significantly upregulated all gene expression by 24 h posttreatment http://www.jimmunol.org/ infected cells, which may have accounted for the high degree of (Fig. 2F, 2G), indicating that the astrocytes were able to induce variability. At 48 h p.i., the transcription factor STAT1, which is a full innate immune response. These results indicate that the IFN phosphorylated upon IFN receptor activation, is not only transla- response is specifically downregulated in SIV-infected astrocytes. tionally upregulated (p = 0.0111) but also has an increase in tyrosine phosphorylation (Y701) (p = 0.0271) (Fig. 1F). Both p- Astrocytes release soluble factors that specifically suppress a STAT1 and STAT1 are induced by ∼75% upon SIV infection, IFN- induction in SIV-infected macrophages indicating that there is a translational and posttranslational up- We found that type I IFN signaling is upregulated in SIV-infected regulation of IFN signaling upon SIV infection. These results in- macrophages in vitro, whereas SIV-infected astrocytes exhibit the by guest on September 27, 2021

FIGURE 1. Type I IFN signaling is upregulated in SIV-infected macrophages. Primary pigtailed macaque MDMs were infected with SIV, and RNA was isolated at 24, 48, and 72 h p.i. (A)Levelsof p27 were measured in the supernatants by ELISA. (B–E) qRT-PCR analysis was done for (B) IFN-b, (C) IFN-a,(D) TRAIL, and (E) MxA. All qRT-PCR data are expressed as a fold induction over uninfected controls. All genes are normalized to 18S rRNA. Data are expressed as the mean of three independent experiments (each done with duplicate technical replicates) from two donor macaques with 95% conﬁdence interval. One-way ANOVA with Bonferroni posttest for multiple comparisons was performed for mRNA comparisons. Signiﬁcant (p , 0.05) pairwise comparisons are shown. (F) Protein lysates were made from UI controls (C) and SIV-infected (I) MDMs at 48 h p.i. and probed for GAPDH, STAT1, and p-STAT1 (Y701) via Western blot. Quantitations were performed on three replicate Western blots normalized to GAPDH. Blot shown is representative of all experiments. An unpaired t test was done to compare uninfected and SIV-infected samples for each protein. 4 CCL2 DISRUPTS TYPE I IFN SIGNALING IN MACROPHAGES Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Type I IFN signaling is downregulated in SIV-infected astrocytes. Primary rhesus astrocytes were infected with SIV. RNA was isolated at 2, 7, 10, 14, and 21 d p.i. (A) p27 levels were measured in the supernatants by ELISA. (B–D) qRT-PCR analysis was done for (B) IFN-a,(C) IFN-b, and (D) MxA. TRAIL mRNA was undetectable. All qRT-PCR data are expressed as a fold induction over uninfected controls. All genes are normalized to 18S rRNA. Data are expressed as the mean of three independent experiments (each done with duplicate technical replicates) with 95% confidence interval. (E) Protein lysates were made at 5, 10, 14, 21, and 25 d p.i. from uninfected controls (C) and SIV-infected (I) primary rhesus astrocytes and probed for GAPDH, STAT1, and p-STAT1 (Y701) via Western blot. Quantitations were performed on four replicate Western blots at 14 d p.i. normalized to GAPDH. Blot shown is representative of all experiments. (F) Astrocytes were stimulated with polyI:C (10 mg/ml), and RNA was isolated at 24, 28, and 72 h p.i. qRT-PCR was done on IFN-a, IFN-b, and Mx. Values are normalized to 18S rRNA and are expressed as a fold induction over untreated controls. (G) Primary rhesus astrocytes were treated with IFN-b (100 U/ml), and RNA was isolated 24, 48, and 72 h p.i. qRT-PCR was done for IFN-a and Mx. Values are normalized to 18S rRNA and are expressed as a fold induction over untreated controls. Data for astrocyte treatments are expressed as the means of duplicate independent experiments (each done with three technical replicates) with 95% confidence interval. (H) Western blot for IRF7 and GAPDH was performed on control (C) and infected (I) astrocytes 14 d p.i. Blot is representative of three replicates. For all mRNA comparisons, one-way ANOVA with Bonferroni posttest for multiple comparisons was performed. Significant (p , 0.05) pairwise comparisons are shown. opposite effect. In brain tissue, however, there is an intermediate coculturing the cells for two reasons: The two primary cell types response. It has been reported that proteins released by astrocytes were from different donor macaques, and obtaining mixed primary have anti-inflammatory effects on stimulated macrophages. Be- glial cultures directly from adult primate brain is extremely costly cause IFN-a is a more potent inflammatory cytokine in the CNS and complicated. Therefore, we studied the effects of astrocytes than IFN-b, we determined if soluble factors released by astro- on macrophages by doing a series of astrocyte supernatant ex- cytes could specifically downregulate IFN-a in SIV-infected change experiments. Supernatants were collected from either macrophages while maintaining the IFN-b response. We opted uninfected or SIV-infected astrocytes and cleared of virus by to perform a series of supernatant exchange experiments instead of ultracentrifugation. Gag p27 ELISAs were done to ensure no in- The Journal of Immunology 5 fectious virus was present (data not shown). All macrophages SIV-infected astrocyte supernatant, yielded a 2.5-fold induction were infected with SIV in normal macrophage media and then of IFN-b mRNA, whereas culturing with uninfected astrocyte cultured with one of three different media conditions: normal supernatants yielded a 1.5-fold induction, although not signifi- macrophage media, uninfected astrocyte supernatant, or SIV- cantly (p . 0.05). IFN-a mRNA expression was significantly infected astrocyte supernatant. Cells were then harvested at 48 h upregulated (p , 0.001) in SIV-infected macrophages cultured p.i., and mRNA expression of IFN-a, IFN-b, MxA, and TRAIL with normal macrophage media (Fig. 3C). IFN-a expression in was measured. macrophages cultured with uninfected astrocyte supernatants also In cells cultured in normal media and infected astrocyte was increased above uninfected although not significantly (p . supernatants, IFN-b mRNA is significantly upregulated upon SIV 0.05). In contrast, SIV-infected macrophages cultured with in- infection compared with that of uninfected controls (p , 0.05 for fected astrocyte supernatant show no induction of IFN-a mRNA both conditions) (Fig. 3B). Normal macrophage media, as well as compared with uninfected controls (p . 0.05) (Fig. 3C). Fur- Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. Astrocytes release soluble factors that specifically suppress IFN-a induction in SIV-infected macrophages. Primary pigtailed MDMs were infected with SIV and then cultured with either normal macrophage media (normal), uninfected astrocyte supernatants [astro (ui)], or infected astrocyte supernatants [astro (inf)]. Cells were harvested at 48 h p.i. (A) Levels of p27 were measured by ELISA in the supernatants. (B–E) RNA was isolated from cells, and qRT-PCR analysis was done for (B) IFN-b,(C) IFN-a,(D) MxA, and (E) TRAIL. Values are expressed as a fold induction over the respective uninfected controls. All values are normalized to 18S rRNA. Graphs represent means of three independent experiments (each done with duplicate technical replicates) with 95% confidence interval. One-way ANOVA with Bonferroni posttest for multiple comparisons was performed for mRNA comparisons. Significance (p , 0.05) or lack thereof is shown for pairwise comparisons discussed in the text. 6 CCL2 DISRUPTS TYPE I IFN SIGNALING IN MACROPHAGES thermore, the induction pattern of downstream ISGs showed that macaque model that activated astrocytes in brain are associated MxA RNA was significantly upregulated in all media conditions with an upregulation of CCL2 protein (43). Although we have (p , 0.05 for normal media, p , 0.001 for both astrocyte con- shown this in vivo, we wanted to confirm it in our astrocytes ditions), with the highest fold inductions (.50-fold) in macro- in vitro. CCL2 protein was measured by ELISA in the super- phages cultured with either type of astrocyte supernatant (Fig. natants of uninfected and SIV-infected astrocytes between days 15 3D). TRAIL mRNA levels were significantly upregulated almost and 20 p.i. We found that CCL2 was upregulated by 55% upon 800-fold in cells cultured in normal macrophage media (p , SIV infection with a trend toward significance (p = 0.0678) (Fig. 0.001), but SIV-infected macrophages cultured with infected or 4A). CCL2 was also measured in uninfected and infected mac- uninfected astrocyte supernatant showed no significant upregula- rophage supernatants, and, although macrophages did upregulate tion (p . 0.05 for both) (Fig. 3E). Taken together, SIV-infected CCL2 upon SIV infection (p = 0.0026), both infected and unin- macrophages cultured with infected astrocyte supernatants most fected macrophages produced at least 10 times less CCL2 than closely resembles the expression pattern in brain, where IFN-a that of astrocytes (Fig. 4A), suggesting that culturing macrophages and TRAIL are suppressed and IFN-b and MxA are significantly with astrocyte supernatants creates an entirely different immuno- induced. These results indicate that SIV-infected astrocytes secrete logical environment than what macrophages are exposed to when soluble factor(s) that, when cultured with SIV-infected macro- cultured alone. phages, lead to a strong suppression of IFN-a and TRAIL mRNA To determine if CCL2 was responsible for differential IFN reg- while not suppressing IFN-b and MxA. ulation, supernatants from SIV-infected astrocytes were incubated in the presence or absence of a neutralizing Ab to CCL2. Primary SIV-

Astrocytes release a higher concentration of CCL2 than infected MDMs were then cultured with either the CCL2 neutralized Downloaded from a macrophages, which can selectively suppress IFN- in or non-neutralized astrocyte supernatant. In macrophages cultured response to SIV infection with infected supernatants without the CCL2 neutralizing Ab, there To identify the soluble factors in the infected astrocyte supernatants was no signiﬁcant induction of IFN-a mRNA upon SIV infection that lead to differential type I IFN expression in SIV-infected (p . 0.05), whereas when cultured with supernatants containing the macrophages, we examined levels of CCL2, which is one of the neutralizing Ab to CCL2, IFN-a mRNA was signiﬁcantly upregu-

major chemokines released by activated astrocytes in brain in lated 7-fold (p , 0.001) upon SIV infection (Fig. 4B). This was not http://www.jimmunol.org/ response to HIV infection (42). We have previously shown in our seen in the presence of a nonspeciﬁc IgG. IFN-b mRNA was up- by guest on September 27, 2021

FIGURE 4. Astrocytes release a higher concentration of CCL2 than macrophages, which can selectively suppress IFN-a in response to SIV infection. (A) Levels of CCL2 were measured in the supernatants of uninfected (UI) and SIV-infected (SIV) primary astrocytes and primary MDMs. Graphs represent mean levels in supernatants from at least three different experiments with SE. An unpaired t test was performed to compare levels of CCL2 in uninfected and SIV-infected supernatants for both astrocytes and macrophages. (B and C) SIV-infected astrocyte supernatants were incubated with either a neutralizing Ab to CCL2 (NA) or a control goat IgG. SIV-infected MDMs were then cultured with either infected astrocyte supernatants (SIV), infected astrocyte supernatants with CCL2 neutralizing Ab (SIV + NA), or infected astrocyte supernatants with control IgG (SIV + IgG). Cells were harvested 48 h p.i., and RNA was isolated. qRT-PCR analysis was done for (B) IFN-a and (C) IFN-b. Graphs represent means of two independent experiments (each done with duplicate technical replicates) with 95% confidence interval. One-way ANOVA with Bonferroni posttest was performed. Significance (p , 0.05) or lack thereof is shown for comparisons discussed in the text. The Journal of Immunology 7 regulated in both media conditions, but only significantly in media ulation of IFN-a (p , 0.01 for TGF-b, p , 0.05 for CCL3) (Fig. without neutralizing Ab (Fig. 4C). These data suggest that CCL2 5E). These data indicate that CCL2 is able to specifically down- production by astrocytes is responsible for the selective suppression modulate mRNA expression of IFN-a and TRAIL in SIV-infected of IFN-a mRNA in SIV-infected macrophages. macrophages while having no effect on the mRNA expression of IFN-b and Mx. Therefore, CCL2 causes a noncanonical IFN Recombinant CCL2 differentially regulates the IFN response signaling cascade in SIV-infected macrophages, where IFN-a and We have shown that CCL2 is highly secreted by astrocytes upon certain ISGs (i.e., TRAIL) are selectively suppressed transcrip- SIV infection and that this chemokine selectively down-modulates tionally. IFN-a mRNA, as its neutralization restores IFN-a expression a upon SIV infection. To examine whether CCL2 alone would re- CCL2-induced IFN- suppression occurs through CCR2 produce this effect, recombinant CCL2 was added to macro- signaling phages in culture to mimic the astrocyte supernatant exchange To examine whether CCL2 exerts its effect through its cognate experiments. Primary macaque MDMs were infected with SIV receptor, CCR2, a small-molecule inhibitor of CCR2, prop- for 6 h and then incubated in the presence or absence of agermanium (100 mM), was used. Propagermanium is an organic recombinant CCL2 (100 ng/ml) in normal macrophage media. small-molecule inhibitor that specifically blocks CCR2 without Treating SIV-infected macrophages with CCL2 significantly stimulating downstream signaling (44). CCL2-treated and un- downregulated both IFN-a and TRAIL mRNA expression (p , treated macrophages were infected in the presence or absence of 0.001 for both genes) (Fig. 5A, 5D). However, neither IFN-b nor inhibitor. Cells were harvested 48 h p.i., and qRT-PCR analysis

MxA mRNA induction in SIV-infected macrophages was affected was performed on RNA for IFN-a and IFN-b. Blocking the CCR2 Downloaded from by treatment with CCL2 (p . 0.05 for both genes) (Fig. 5B, 5C). receptor in the presence of CCL2 was able to restore IFN-a in- To test the speciﬁcity of the CCL2 effect, primary macrophages duction levels in SIV-infected macrophages (p , 0.01), whereas were also treated with CCL3, which is another chemokine, and neither the presence of CCL2 nor the blocking of CCR2 affected TGF-b (10 ng/ml and 0.2 ng/ml, respectively), which is a cytokine mRNA levels of IFN-b (Fig. 6A, 6B). These data indicate that that astrocytes are known to produce (37). Infection of cells CCL2 mediates the selective suppression of IFN-a in SIV-infected

treated with either of these proteins resulted in significant upreg- macrophages via a CCR2-dependent mechanism. http://www.jimmunol.org/ by guest on September 27, 2021 FIGURE 5. Recombinant CCL2 differentially regulates the IFN response. (A–D) Primary MDMs were infected with SIV and then either treated with CCL2 (CCL2) or left untreated (UT). Cells were harvested at 48 h p.i., and RNA was isolated. qRT-PCR analysis was done for (A) IFN-a,(B) IFN- b,(C) MxA, and (D) TRAIL. Data represent means of three independent experiments (each done with two technical replicates) with 95% confidence interval. (E) Primary MDMs were infected with SIV and then treated with either TGF-b or CCL3 (0.2 ng/ml and 10 ng/ml respectively) or left untreated (UT). Cells were harvested at 48 h p.i., and RNA was isolated. qRT-PCR analysis was done for IFN-a. Data represent means of two independent experiments (each done with two technical replicates) with 95% confidence interval. Two-way ANOVAwith Bonferroni posttest for multiple comparisons was performed. Significance (p , 0.05) or lack thereof is shown for comparisons discussed in the text. 8 CCL2 DISRUPTS TYPE I IFN SIGNALING IN MACROPHAGES

FIGURE 6. CCL2-induced IFN-a suppression occurs through CCR2 signaling. (A and B) SIV-infected primary MDMs were treated with either propagermanium (prop), CCL2 (CCL2), CCL2 plus propagermanium (CCL2 + prop), or left untreated (UT). Cells were harvested 48 h p.i., and RNA was isolated. qRT-PCR analysis was done for (A) IFN-a or (B) IFN-b. Data represent means of three independent experiments (each done with two technical replicates) with 95% conﬁdence interval. One-way ANOVA with Bonferroni posttest for multiple comparisons was performed on the SIV-infected group, and selected pairwise comparisons are shown. Results were considered signiﬁcant with p , 0.05. Downloaded from

Discussion there is no signiﬁcant induction in peripheral tissues such as the In this report, we identiﬁed the cellular pathway that leads to the lung (J. Clements, unpublished observations). We have previously noncanonical type I IFN signaling pattern seen during acute SIV found that suppression of IFN-a mRNA occurs in the brain during infection in the macaque brain. We show that SIV-infected mac- acute infection, but not in lung (2). Thus, the CNS, which contains

rophages cultured with supernatants from SIV-infected astrocytes a high concentration of CCL2, exhibits differential type I IFN http://www.jimmunol.org/ specifically suppress IFN-a and TRAIL mRNA while maintaining regulation, whereas tissues in the periphery such as the lung, an induction of IFN-b and MxA. This effect was due to the high which are not exposed to as much CCL2, do not. These results concentration of CCL2 that infected astrocytes produce, as neu- provide valuable insight into how the compartmentalization of tralizing CCL2 in the infected astrocyte supernatants ameliorated different tissues and cell types results in an entirely different this selective suppression of IFN-a and TRAIL. The addition of immunological response. recombinant CCL2 to SIV-infected macrophages reproduced this Why this differential regulation of the IFN signaling pathway selective suppression, and this was mediated through the CCR2 occurs is most likely due to type of tissue in which infection occurs. receptor. This is a novel and important discovery that shows how Astrocytes are cells that are exclusively found in the brain and are CCR2 signaling can communicate with the type I IFN pathway to potent producers of CCL2 upon HIV infection (42, 47). Macro- by guest on September 27, 2021 alter the canonical IFN response. phages and microglia are strong producers of type I IFNs in re- The complete suppression of the type I IFN response in SIV- sponse to viral infection in the CNS, but each IFN has very infected astrocytes is very striking. Because astrocytes are able different effects. IFN-a has known neurotoxic and neuroinflam- to induce the IFN response in polyI:C-treated and IFN-b–treated matory effects. Not only is it associated with HIV dementia and cells, this suppression of signaling in astrocytes is specific to SIV genetic diseases such as Aicardi-Goutie`res syndrome and Cree infection. We report that there is a deficiency in IRF7 mRNA and encephalitis, which are characterized by neurodegeneration and protein induction upon SIV infection, which may be responsible ongoing inflammatory processes, but also hepatitis C patients on for the inability to produce IFN-a and IFN-b. The exact intra- IFN-a therapy have been known to suffer from depression, seiz- cellular sensor that recognizes HIV and SIV in astrocytes is cur- ures, and EEG changes (4–6, 8, 10). IFN-b, however, has been rently unknown, although one group has suggested the mannose associated with the secretion of anti-inflammatory cytokines and receptor as playing a role (45). Therefore, more work is needed to neurotropic growth factors (9, 48). Therefore, the high level of determine which upstream innate intracellular regulators are being CCL2 production by astrocytes in the CNS may be an evolu- affected during infection that leads to lack of IRF7 activation. It is tionary mechanism that limits the production of proteins that lead already known that astrocytes contain restrictive factors that se- to neuronal death, such as IFN-a and TRAIL, while allowing the verely impair the kinetics of SIV and HIV replication (39, 40). It is induction of cytokines that can provide protection against patho- therefore possible that these restrictive factors may somehow gens without the destructive effects, such as IFN-b and MxA. This prevent activation of the innate IFN response by interfering with balance is extremely important in the CNS, as it is an organ the production of viral intermediates that would otherwise be composed of nonrenewable neurons. However, in the periphery, immediately recognized by PRRs. where cell turnover is particularly high and apoptosis can serve Although macrophages do upregulate CCL2 protein upon SIV a protective purpose rather than a permanent loss of nonrenewable infection, the amount of CCL2 is 10 times less than the amount cells, this fine-tuned balance between inflammatory versus anti- produced by infected astrocytes. This suggests that immunological inflammatory cytokine production is not as vital. signaling is affected by not only the type of soluble factors that are The immunomodulatory and protective effects of CCL2 on cells present in a particular microenvironment but also the concentration. in the CNS have been well characterized. CCL2 has been reported The suppressive effects that high concentrations of CCL2 have on to protect neurons and astrocytes from HIV-tat–induced and N- IFN-a mRNA that we see in vitro are consistent with what we see methyl-D-aspartate–induced apoptosis (30, 49). Furthermore, the in vivo in our macaque model. During acute SIV infection, there is treatment of microglia and neurons with CCL2 does not activate ∼10 times more CCL2 protein production in the cerebrospinal an inflammatory response, nor does it induce apoptosis, indicating fluid than there is in the plasma (43). Furthermore, there is an that this chemokine does not inflict deleterious inflammatory almost 100-fold induction of CCL2 mRNA in brain (46), whereas effects (50). In addition, CCR2 deficiency in microglia has been The Journal of Immunology 9 reported to accelerate disease progression in a mouse model of Disclosures Alzheimer’s disease (51). Our findings that CCL2 suppresses The authors have no financial conflicts of interest. signaling pathways leading to IFN-a, which is a known inflammatory modulator in the CNS, coincides with evidence in the literature of its neuroprotective role. References 1. Kawai, T., and S. Akira. 2007. 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