Journal of Autoimmunity xxx (2017) 1e7 Contents lists available at ScienceDirect Journal of Autoimmunity journal homepage: www.elsevier.com/locate/jautimm Short communication MicroRNA-302d targets IRF9 to regulate the IFN-induced gene expression in SLE Siobhan Smith a, Thilini Fernando b, c, Pei Wen Wu b, c, Jane Seo b, c, Joan Ní Gabhann a, Olga Piskareva a, Eoghan McCarthy d, Donough Howard d, Paul O'Connell e, Richard Conway e, Phil Gallagher e, Eamonn Molloy e, Raymond L. Stallings a, Grainne Kearns c, Lindsy Forbess b, Mariko Ishimori b, Swamy Venuturupalli b, * Daniel Wallace b, Michael Weisman b, Caroline A. Jefferies a, b, c, a Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin 2, Ireland b Division of Rheumatology, Department of Medicine, Cedars-Sinai Medical Centre, 8700 Beverly Blvd, Los Angeles, CA 90048, USA c Department of Biomedical Sciences, Cedars-Sinai Medical Centre, 8700 Beverly Blvd, Los Angeles, CA 90048, USA d Department of Rheumatology, Beaumont Hospital, Dublin 9, Ireland e Department of Rheumatology, St. Vincent's University Hospital, Dublin 4, Ireland article info abstract Article history: Systemic lupus erythematosus (SLE) is a complex disease targeting multiple organs as a result of over- Received 31 January 2017 activation of the type I interferon (IFN) system, a feature currently being targeted by multiple biologic Received in revised form therapies against IFN-a. We have identified an estrogen-regulated microRNA, miR-302d, whose 2 March 2017 expression is decreased in SLE patient monocytes and identify its target as interferon regulatory factor Accepted 6 March 2017 (IRF)-9, a critical component of the transcriptional complex that regulates expression of interferon- Available online xxx stimulated genes (ISGs). In keeping with the reduced expression of miR-302d in SLE patient mono- cytes, IRF9 levels were increased, as was expression of a number of ISGs including MX1 and OAS1. In vivo Keywords: fl SLE evaluation revealed that miR-302d protects against pristane-induced in ammation in mice by targeting MicroRNA IRF9 and hence ISG expression. Importantly, patients with enhanced disease activity have markedly IFN signalling reduced expression of miR-302d and enhanced IRF9 and ISG expression, with miR-302d negatively IFN-stimulated genes correlating with IFN score. Together these findings identify miR-302d as a key regulator of type I IFN driven gene expression via its ability to target IRF9 and regulate ISG expression, underscoring the importance of non-coding RNA in regulating the IFN pathway in SLE. © 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction a signalling cascade that results in the activation of two cytoplasmic kinases JAK1 and TYK2, which subsequently phosphorylate the Systemic lupus erythematosus (SLE) is a chronic autoimmune associated transcription factors STAT1 and STAT2. Once phosphor- disease characterised by a wide variety of immunological defects ylated, STAT1 and STAT2 dimerize and interact with IRF9 to form including enhanced expression of type I interferon (IFN)-stimu- the transcriptionally active complex, ISGF3, which binds to IFN- lated genes (ISGs) as a result of increased circulating levels of the stimulated response elements (ISRE) in the promoter region of antiviral cytokines IFN-alpha and IFN-beta (IFN-a and -b) [1]. IFN-inducible genes (reviewed in Ref. [3]). Intraperitoneal injection Elevated IFN-a correlates with disease severity, flare and tissue of the hydrocarbon oil 2,6,10,14-tetramethylpentadecane (TMPD; involvement (specifically in the skin, kidney and central nervous also known as pristane) in mouse strains including C57BL/6 or Balb/ system) [2]. Binding of IFN-a/b to the IFN receptor (IFNAR) activates c mice has been used to induce a TLR7-dependent induction of type I IFN and sustained expression of interferon stimulated genes as early as 2 weeks post-pristane injection [4,5]. Although dendritic * Corresponding author. Division of Rheumatology, Department of Medicine and cells are the major producers of IFN, it is Ly6Chi monocytes which Department of Biomedical Sciences, Cedars-Sinai Medical Centre, 8700 Beverly are the predominant source of IFN-a and -b in the pristane model of Blvd, Los Angeles, CA 90048, USA. E-mail address: [email protected] (C.A. Jefferies). lupus, suggesting monocytes to be of importance in the http://dx.doi.org/10.1016/j.jaut.2017.03.003 0896-8411/© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: S. Smith, et al., MicroRNA-302d targets IRF9 to regulate the IFN-induced gene expression in SLE, Journal of Autoimmunity (2017), http://dx.doi.org/10.1016/j.jaut.2017.03.003 2 S. Smith et al. / Journal of Autoimmunity xxx (2017) 1e7 interferonopathy observed in SLE [6e8]. Both IRF9 and IRF5 have 2.2. Isolation of PBMCs and cellular subsets independently been shown to be required for IFN-driven responses in pristane-induced ISG expression and autoimmunity [5,9]. Peripheral blood mononuclear cells (PBMCs) were separated With 90% of SLE patients being female, enhanced signalling from whole blood by density-gradient centrifugation with Ficoll- þ through estrogen receptor-alpha (ER-a) has been proposed to help Paque Plus (GE Healthcare). CD14 monocytes were purified from þ explain the strong female prevalence of SLE [10], with many studies fresh PBMCs by positive selection using magnetic anti-CD14 beads in both mouse models of lupus and ex vivo in SLE patient cells (Miltenyi Biotec and STEMCELL Technologies) according to manu- supporting this [11e16]. In addition, we have recently reported that facturers' protocol. levels of ER-a (ESR1) expression are elevated in SLE patient monocytes, with enhanced expression corresponding with 2.3. Cell culture increased sensitivity of SLE patient monocytes to estrogen stimu- lation in driving the expression of IL-23 and IFN-b, for example [11]. Purified monocytes were cultured in phenol redefree RPMI Cross-talk between estrogen-driven signalling and the IFN system 1640 medium supplemented with 10% charcoalestripped foetal is evidenced by the ability of IFN-a to drive ESR1 upregulation in calf serum and 100 mg/ml of penicillin/streptomycin. Sterile splenic mouse cells, with enhanced levels of ISGs and ESR1-driven ethanol-soluble 17b-estradiol and MPP (Sigma) were prepared genes observed in SLE-prone (NZB Â NZW) F1 female mice fresh for each experiment. Cells were stimulated for 6 h with sterile compared to their male counterparts [17]. water-soluble type I IFN (Immunotools) at 1000 IU/ml. Regarding the potential that estrogen-regulated microRNAs (miRs) are involved in either SLE development or pathogenesis, 2.4. MicroRNA mimic and inhibitor transfection female lupus prone-NZB/W (F1) mice have increased expression of lupus-linked miRNAs including miR-32, miR-155, miR-127 and the MicroRNA oligonucleotides were obtained from Dharmacon. miR-182-96-183 cluster compared to their male counterparts [18]. Negative controls were based on the sequences of miRNA in Cae- Moreover, X-linked miRNAs - miR-503, miR-188-3p, miR-421 and norhabditis elegans (cel-miR-67). Reverse transfection of human þ miR-98 - were reported to be overexpressed in CD4 T cells from primary monocytes was performed using Metafectene SI trans- female SLE patients compared to male patients [19], potentially fection reagent according to the manufacturers protocol. Cells were contributing to the sex differences observed in SLE development. To harvested for analysis 48e72 h after transfection. date however no association between estrogen-regulated micro- RNAs in SLE and IFN-driven gene expression has been shown. 2.5. Real-time quantitative polymerase chain reaction (RT-qPCR) In this study, we have identified miR-302d as an estrogen- analysis regulated miRNA which is downregulated in the immune cells of patients with SLE. Bioinformatic analysis identified interferon RNA was extracted from cell cultures using TRIzol reagent regulatory factor 9 (IRF9), a transcription factor which plays a key (Sigma) according to manufacturer's protocol. Expression of >800 ® role in promoting IFN-a-mediated gene expression, as a target miRNAs was assessed by Nanostring Technologies using nCounter gene. We validated the miR-302d binding site in the 30 untranslated miRNA Expression Assays. To validate the results, miR-302d region (UTR) of IRF9 and demonstrated augmentation of IRF9 levels expression was analyzed using the miScript primer assay with transfection of a miR-302d mimic, accompanied by enhanced (MS00003920 Qiagen) and miScript SYBR Green PCR kit (Qiagen), ISG expression. We demonstrate that in vivo administration of miR- with normalisation to the U6 small nuclear RNA (U6 snRNA). RNA 302d decreases IRF9 levels both in the peritoneum and systemically was reverse transcribed using the Tetro cDNA synthesis kit (Med- and that this results in reduced expression of MX1 and ISG15. ical Supply Company) according to the manufacturer's recom- Translating this to human SLE, we have found that not only is IRF9 mendations. Expression of mRNA was determined using expression elevated in SLE monocytes, but that its expression is appropriate primers (Supplementary Table 2) with the Sensifast associated with enhanced levels of ISGs at baseline in SLE patient Real-Time PCR Kit (Medical Supply Company) according to the peripheral blood mononuclear cells (PBMCs). Importantly, reduced manufacturer's recommendations. Data were analyzed using the miR-302d expression in patient PBMCs is associated with more DDCt comparative quantification method following normalisation active disease, underscoring the importance of miR-302d expres- to 18sRNA [21]. sion in maintaining a balance in the IFN system via its ability to regulate IRF9 expression. 2.6. Luciferase reporter assay The putative miR-302d target sequence in the 30UTR of human 2.