Regulation of IFN-λ1 Promoter Activity (IFN- λ1/IL-29) in Human Airway Epithelial Cells This information is current as Rachael Siegel, Joyce Eskdale and Grant Gallagher of September 27, 2021. J Immunol 2011; 187:5636-5644; Prepublished online 4 November 2011; doi: 10.4049/jimmunol.1003988 http://www.jimmunol.org/content/187/11/5636 Downloaded from References This article cites 48 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/187/11/5636.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • 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 by guest on September 27, 2021 *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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Regulation of IFN-l1 Promoter Activity (IFN-l1/IL-29) in Human Airway Epithelial Cells Rachael Siegel, Joyce Eskdale, and Grant Gallagher The type III (l) IFNs (IFN-l1, IFN-l2, and IFN-l3) and their receptor are the most recently discovered IFN family. They are induced by viruses and mediate antiviral activity, but type III IFNs have an important, specific functional niche at the immune/ epithelial interface, as well as in the regulation of Th2 cytokines. Their expression appears diminished in bronchial epithelial cells of rhinovirus-infected asthmatic individuals. We investigated the regulation of IFN-l1 expression in human airway epithelial cells using reporter genes analysis, chromatin immunoprecipitation, small interfering RNA knockdown, and DNase footprinting. In this article, we define the c-REL/p65 NF-kB heterodimer and IRF-1 as key transcriptional activators and ZEB1, B lymphocyte- induced maturation protein 1, and the p50 NF-kB homodimer as key repressors of the IFN-l1 gene. We further show that ZEB1 selectively regulates type III IFNs. To our knowledge, this study presents the first characterization of any type III IFN Downloaded from promoter in its native context and conformation in epithelial cells and can now be applied to understanding pathogenic dysreg- ulation of IFN-l1 in human disease. The Journal of Immunology, 2011, 187: 5636–5644. he type III (l) IFNs 1, 2, and 3 (IFN-l1, IFN-l2, and IFN- on distal NF-kB sites within the promoter (21137, 21182 bp) (7). l3), also known as IL-29, IL-28A, and IL-28B, respec- These data suggested a regulation that may be both cell type and T tively, are the most recently defined IFN ligands in stimulation specific, perhaps necessary to maintain efficient con- http://www.jimmunol.org/ humans (1, 2). In addition to antiviral properties, this class of IFNs trol over IFN-l expression and its role in the immune response. has important but poorly characterized functions in innate and The tightly targeted nature of the IFN-l response is distinct from adaptive immunity. Type III IFNs are four-helical-bundle cyto- the more ubiquitous secretion and response to type I IFNs. Ex- kines that are distantly related to both the IL-10 family and the pression of IFN-l is restricted mainly to plasmacytoid dendritic type I IFN family (3). In humans, the three IFN-l proteins are cells and epithelial cells (8, 9). Furthermore, expression of the encoded by genes on chromosome 19q13; however, IFN-l1 is not IFN-l receptor (in which both chains are distinct from those used functionally conserved in mice. Although all three gene products in either the type I or type II IFNRs) is almost completely re- share a high degree of amino acid similarity, the IFN-l2 and IFN- stricted to immune cells and epithelial cells (9–12). Thus, the l3 proteins are .95% identical and may have arisen from a recent restricted expression pattern of the IFN-l ligands and their re- by guest on September 27, 2021 gene-duplication event (1–4). ceptor confers tight control on IFN-l responsiveness, in contrast Studies of IFN-l gene regulation have focused on comparing to the situation with the ubiquitous type I IFNs. it with that of the type I IFN genes (5, 6). Onoguchi et al. (5) Although the type I and type III IFNs use distinct receptor demonstrated dependence on NF-kB and IRF sites within the first complexes, their downstream-signaling events are very similar. 300 bases of the IFN-l1 promoter following infection of trans- Type III IFNs primarily trigger STAT phosphorylation; pSTAT1 formed cells with Newcastle disease virus. Osterlund et al. (6) homodimers and pSTAT1/2 heterodimers are both formed, and extended these findings to suggest that IFN-l1, like IFN-b,is target-gene promoter g IFN activation site (GAS) element and IFN- regulated by IRF-3, whereas IFN-l2/3 are more dependent upon sensitive response element (ISRE) are both activated (1). IFN- IRF7, similar to IFN-a. In addition, a recent study using bacterial stimulated genes, such as myxovirus resistance protein (MX1) infection of human monocyte-derived dendritic cells (MDDC) and 29-59-oligoadenylate synthetase (OAS), are IFN-l responsive showed that, in this case, IFN-l1 activation was more dependent (1, 13–15); consequently, IFN-ls induce biological activities, in- cluding antiviral, antiproliferative, and proapoptotic effects that are similar to those of the type I IFNs (16). The antiviral properties Genetic Immunology Laboratory, HUMIGEN LLC, Institute for Genetic Immunol- ogy, Genesis Biotechnology Group, Hamilton, NJ 08690 of the three IFN-ls vary (17) but are generally held to be weaker Received for publication December 6, 2010. Accepted for publication September 30, than those of the type I ligands (18). 2011. The IFN-ls were recently shown to have immunoregulatory R.S. designed and executed experiments, analyzed data, and wrote, revised, and functions (19, 20), particularly an ability to downregulate human finalized the manuscript. J.E. designed and executed experiments, analyzed data, Th2 responses. These studies showed that IFN-l1 acts to limit Th2 and finalized the manuscript. G.G. designed experiments, analyzed data, and wrote and finalized the manuscript. polarization and the secretion of the Th2 cytokines IL-4, IL-5, and + Address correspondence and reprint requests to Prof. Grant Gallagher, Genetic Im- IL-13. These effects can be direct on CD4 T cells or indirect, via munology Laboratory, HUMIGEN, Institute for Genetic Immunology, 2439 Kuser MDDC. Further, IFN-l1 secretion is itself Th2 cytokine respon- Road, Hamilton, NJ 08690. E-mail address: [email protected] sive (11, 12, 21–23). Recent studies also showed that IFN-l1 Abbreviations used in this article: BLIMP-1, B lymphocyte-induced maturation pro- expression is reduced in alveolar macrophages and bronchial ep- tein 1; ChIP, chromatin immunoprecipitation; GAS, g IFN activation site; ISRE, IFN- sensitive response element; MDDC, monocyte-derived dendritic cell; MX1, myxo- ithelial cells of rhinovirus-infected asthmatic individuals (24, 25). virus resistance protein; NT, nontargeting; poly I:C, polyinosinic-polycytidylic acid; Two key contributing pathologies in asthma are the upregulation qRT-PCR, quantitative RT-PCR; RV1B, human rhinovirus 1B; siRNA, small inter- of Th2 responses and remodeling of the airway epithelial cells fering RNA; TSS, translation start site. (26). Thus, the observed lack of a key regulator (IFN-l1) may be Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 critical in asthmatic individuals, because it normally would be www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003988 The Journal of Immunology 5637 produced primarily by plasmacytoid dendritic cells and epithelial Small interfering RNA knockdown cells to both promote viral responsiveness and limit Th2 responses SmartPool small interfering RNA (siRNA) to target NF-kB1 (p50), RELA (8–11). (p65), ZEB1, B lymphocyte-induced maturation protein 1 (BLIMP-1), Studies of promoter function continue to shed light on the role GAPDH, or control nontargeting (NT) siRNA were purchased from of immune-system components in health and disease (27–30). To Thermo-Scientific. These were transfected into BEAS-2B or A549 cells begin to explore the nature of IFN-l1 production by airway epi- using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. siGLO (Thermo-Scientific) was used to optimize transfection thelial cells, we characterized the regulation of IFN-l1 tran- efficiency (87%; data not shown). At 24 h posttransfection, cells were scription in a model of human airway viral infection. reseeded at 2.0 3 105 cells/ml, and poly I:C stimulation or viral infection was initiated 12 h after reseeding. Supernatants, protein from whole-cell extracts, and total RNA were harvested following 0, 3, 4.5, 8, 24, or 32 h Materials and Methods of poly I:C stimulation or human rhinovirus 1B (RV1B) infection. RV1B Reporter assays (American Type Culture Collection) infection was performed at a multi- plicity of infection of 1, where the virus was incubated with the cells 9 l Four kilobases 5 of the IFN- 1 translation start site (TSS) were PCR grown in RPMI 1640 containing 2% FBS. Quantitative RT-PCR (qRT- amplified, sequence-verified, and cloned upstream of the firefly luciferase PCR) was then used to analyze the mRNA levels of genes of interest, gene in the pGL4.10 reporter vector (Promega, Madison WI).