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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 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 27, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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.

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