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The PYHIN Protein P205 Regulates the Inflammasome by Controlling Asc Expression

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The PYHIN Protein P205 Regulates the Inflammasome by Controlling Asc Expression The PYHIN Protein p205 Regulates the Inflammasome by Controlling Asc Expression This information is current as Sreya Ghosh, Christina Wallerath, Sergio Covarrubias, Veit of September 29, 2021. Hornung, Susan Carpenter and Katherine A. Fitzgerald J Immunol 2017; 199:3249-3260; Prepublished online 20 September 2017; doi: 10.4049/jimmunol.1700823 http://www.jimmunol.org/content/199/9/3249 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/09/20/jimmunol.170082 Material 3.DCSupplemental http://www.jimmunol.org/ References This article cites 58 articles, 22 of which you can access for free at: http://www.jimmunol.org/content/199/9/3249.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 29, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average 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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology The PYHIN Protein p205 Regulates the Inflammasome by Controlling Asc Expression Sreya Ghosh,* Christina Wallerath,†,‡ Sergio Covarrubias,x Veit Hornung,†,‡ Susan Carpenter,x and Katherine A. Fitzgerald* Members of the IFN-inducible PYHIN protein family, such as absent in melanoma-2 and IFN-g–inducible protein (IFI)16, bind dsDNA and form caspase-1–activating inflammasomes that are important in immunity to cytosolic bacteria, DNA viruses, or HIV. IFI16 has also been shown to regulate transcription of type I IFNs during HSV infection. The role of other members of the PYHIN protein family in the regulation of immune responses is much less clear. In this study, we identified an immune-regulatory function for a member of the murine PYHIN protein family, p205 (also called Ifi205). Examination of immune responses induced by dsDNA and other microbial ligands in bone marrow–derived macrophages lacking p205 revealed that inflammasome activation by dsDNA, as well as ligands that engage the NLRP3 inflammasome, was severely compromised in these cells. Further analysis Downloaded from revealed that p205-knockdown cells showed reduced expression of apoptosis-associated speck-like molecule containing CARD domain (Asc) at the protein and RNA levels. p205 knockdown resulted in reduced binding of actively transcribing RNA poly- merase II to the endogenous Asc gene, resulting in decreased transcription and processing of Asc pre-mRNA. Deletion of p205 in B16 melanoma cells using CRISPR/Cas9 showed a similar loss of Asc expression. Ectopic expression of p205 induced expression of an Asc promoter–luciferase reporter gene. Together, these findings suggest that p205 controls expression of Asc mRNA to regulate inflammasome responses. These findings expand on our understanding of immune-regulatory roles for the PYHIN protein http://www.jimmunol.org/ family. The Journal of Immunology, 2017, 199: 3249–3260. oreign nucleic acids (NAs) play a critical role in initiating form of cell death called pyroptosis (10). Although most inflam- innate and adaptive immune responses. NA sensors expressed masomes are composed of members of the NLR family, the dsDNA- F in distinct cellular compartments survey the extracellular and activated inflammasome is formed following dsDNA binding to a intracellular environment for signs of infection and initiate immune PYHIN protein, AIM2. Work from several laboratories, including defenses against bacterial, viral, and eukaryotic pathogens (1). NA our own, has defined AIM2 as a cytosolic DNA- binding innate sensors include RNA sensors, such as TLR3, TLR7/8, and RIG-I/ immune receptor (7, 11–13). AIM2 binds pathogen-derived dsDNA by guest on September 29, 2021 MDA5 (2–4), as well as DNA sensors, such as TLR9, cGAS, absent that accumulates in the cytosol during infection with DNA viruses or in melanoma-2 (AIM2), and IFN-g–inducible protein (IFI)16 (5–8). cytosolic bacterial pathogens (14, 15). In some instances, AIM2 can In addition, recognition of foreign NAs, especially dsDNA, leads also recognize host dsDNA that gains access to the cytosol leading to assembly of an inflammasome, a large caspase-1–activating multi- to autoinflammation (16). The related PYHIN protein IFI16 also protein complex that controls the proteolytic processing and release forms an inflammasome during infection with Kaposi’s sarcoma of IL-1b (9). Inflammasome activation also results in a proinflammatory herpesvirus and HIV-1 (17, 18). The PYHIN proteins were first characterized as a family of IFN- inducible proteins that are predominantly nuclear localized (19). *Program in Innate Immunity, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605; †Gene Center, Ludwig Maximilian University of PYHINs are constitutively expressed in different hematopoietic Munich, Munich 81377, Germany; ‡Department of Biochemistry, Ludwig Maximilian cell types, although most members of this family are also induc- University of Munich, Munich 81377, Germany; and xDepartment of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA 95064 ible by type I IFN in nonhematopoietic cells (19). Phylogenetic Received for publication June 7, 2017. Accepted for publication August 22, 2017. analysis of the mammalian PYHIN proteins, also called the AIM2- like receptors, show strong evolutionary and functional diversity (20, This work was supported by National Institutes of Health Grants AI093752, AI083713, and AI067497. 21). The murine PYHIN locus has undergone extensive gene du- Address correspondence and reprint requests to Dr. Katherine A. Fitzgerald, Program plication, with 13 members encoded in the murine genome in contrast in Innate Immunity, Department of Medicine, University of Massachusetts Medical to the human gene locus with 5 genes, including MNDA, PYHIN1, School, LRB, Room 308, 364 Plantation Street, Worcester, MA 01605. E-mail ad- dress: kate.fi[email protected] POP3, IFI16, and AIM2 (19, 22). PYHIN proteins have been impli- The online version of this article contains supplemental material. cated in a wide range of cellular processes, including transcription, tumor suppression, cell cycle, cell growth, differentiation, and cell Abbreviations used in this article: AIM2, absent in melanoma-2; Asc, apoptosis-associated speck-like molecule containing CARD domain; BMDM, bone marrow–derived death (19). The majority of the PYHIN proteins share the same macrophage; CDS, coding DNA sequence; ChIP, chromatin immunoprecipitation; structural domains. They contain an N-terminal a-helical domain, CTD, C-terminal domain; GFP shRNA, shRNA sequence targeting GFP; HA, hem- agglutinin; iBMDM, immortalized BMDM; IFI, IFN-g–inducible protein; ISD, IFN known as the pyrin domain, which is capable of homotypic stimulatory DNA; KD, knockdown; KO, knockout; LDH, lactate dehydrogenase; protein–protein interactions, and one or more HIN200 domains in NA, nucleic acid; pdAdT, poly-dAdT; poly I:C, polyinosinic-polycytidylic acid; their C terminus, with the exception of murine p208 and human qPCR, quantitative PCR; RNA Pol II, RNA polymerase II; serine-2P RNA Pol II, RNA Pol II phosphorylated at serine 2; serine-5P RNA Pol II, RNA Pol II phos- POP3, which contain only a pyrin domain, and murine p202, phorylated at serine 5; shRNA, short hairpin RNA; SV, Sendai virus; 39UTR, 39 which contains only two HIN200 domains (19, 20). Most PYHIN untranslated region. proteins contain a nuclear localization signal in their N terminus Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 that can be monopartite, bipartite, or both. Some family members www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700823 3250 p205 REGULATES INFLAMMASOME ACTIVATION also contain a nuclear export sequence that enables them to shuttle dehydrogenase (LDH), which is released into the supernatant upon cell lysis, between the nucleus and the cytosol. AIM2 is the most conserved using a CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) kit. family member. Unlike the other PYHINs, AIM2 is localized in Triton-X (10%) was added to the cells as a representation of 100% cell death. the cytosol. Phylogenetic analysis indicates that, with the exception Short hairpin RNA–mediated silencing of AIM2, there is a complete lack of orthology among mammalian The short hairpin RNA (shRNA) sequences targeting p205 were cloned into AIM2-like receptors (20, 21). a lentiviral pLKO.1 TRC cloning vector. Two separate shRNA sequences Although the role of AIM2 and IFI16 in dsDNA recognition and used for knocking down p205 targeted the coding DNA sequence (CDS) immune activation has been well established, the role of other (TRCN0000095887) or the 39 untranslated region (39UTR) (TRCN0000095884). members of the PYHIN protein family, especially those in the HEK 293T cells were transfected with 4 mg of shRNA along with 3 mgof pSPAX (gag/pol) and 1 mg of pMD2 (VSV-G) plasmids for production of mouse, remains unclear.
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