Cutting Edge: Intracellular IFN-β and Distinct Type I IFN Expression Patterns in Circulating Systemic Lupus Erythematosus B Cells This information is current as of October 4, 2021. Jennie A. Hamilton, Qi Wu, PingAr Yang, Bao Luo, Shanrun Liu, Jun Li, Alexa L. Mattheyses, Ignacio Sanz, W. Winn Chatham, Hui-Chen Hsu and John D. Mountz J Immunol published online 10 September 2018 http://www.jimmunol.org/content/early/2018/09/07/jimmun Downloaded from ol.1800791 Supplementary http://www.jimmunol.org/content/suppl/2018/09/07/jimmunol.180079 Material 1.DCSupplemental 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 by guest on October 4, 2021 • 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 © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 10, 2018, doi:10.4049/jimmunol.1800791 Cutting Edge: Intracellular IFN-b and Distinct Type I IFN Expression Patterns in Circulating Systemic Lupus Erythematosus B Cells Jennie A. Hamilton,* Qi Wu,* PingAr Yang,* Bao Luo,* Shanrun Liu,* † ‡ Jun Li,* Alexa L. Mattheyses, Ignacio Sanz,x W. Winn Chatham,* Hui-Chen Hsu,* and John D. Mountz*, In systemic lupus erythematosus (SLE), type I IFNs pro- different immune cell populations (3–7), but cell-specific mote induction of type I IFN–stimulated genes (ISG) expression patterns and roles of distinct type I IFNs in SLE, and can drive B cells to produce autoantibodies. Little especially high-affinity IFN-b, remain elusive (8), largely is known about the expression of distinct type I IFNs because of their low levels of transcription and circulation (9). in lupus, particularly high-affinity IFN-b. Single-cell In autoimmune mice, IFNaR1 deficiency ameliorates ger- Downloaded from analyses of transitional B cells isolated from SLE pa- minal center and autoantibody development (10, 11). Although a previous study in NZB mice reported no difference in anti- tients revealed distinct B cell subpopulations, including ⁄ chromatin Abs or renal disease in Ifnb–– mice (12), these type I IFN producers, IFN responders, and mixed IFN b producer/responder clusters. Anti-Ig plus TLR3 stim- findings have been challenged by reports that IFN- is elevated and dysregulated in SLE (13, 14) as well as the identification of ulation of SLE B cells induced release of bioactive type a http://www.jimmunol.org/ I IFNs that could stimulate HEK-Blue cells. Increased distinct IFN signatures not restricted to IFN- (15). Serum detection of IFN-b was recently associated with disease flares, levels of IFN-b were detected in circulating B cells particularly in African American (AA) patients, a population from SLE patients compared with controls and were with increased disease prevalence, severity, and robust type I significantly higher in African American patients with IFN dysregulation (14, 16). Autocrine IFN-b signaling has renal disease and in patients with autoantibodies. To- been identified as a mechanism of type I IFN dysregulation in gether, the results identify type I IFN–producing and SLE mesenchymal stem cells (13), and B cells have also been –responding subpopulations within the SLE B cell shown to produce type I IFNs in SLE and other diseases (9, 17, compartment and suggest that some patients may ben- 18). In this study, we examined expression patterns of type I by guest on October 4, 2021 efit from specific targeting of IFN-b. The Journal of IFNs and their target type I IFN–stimulated genes (ISGs) Immunology, 2018, 201: 000–000. in circulating B cells and identified a potential role for B cell– associated IFN-b in SLE. ctivation of the type I IFNs, consisting of 13 IFN-a subtypes and one high-affinity IFN-b subtype, is Materials and Methods A highly associated with the development of systemic Clinical samples lupus erythematosus (SLE) as well as clinical disease mani- All SLE subjects met the American College of Rheumatology 1997 re- festations (1). Type I IFNs can be produced by most cell vised criteria for SLE (19) and were recruited from the University of types, although their activity in SLE is most often measured Alabama at Birmingham (UAB) Lupus Clinic. PBMCs were isolated by indirectly using the presence of specific type I IFN–inducible density gradient centrifugation (Lymphoprep/SepMate; STEMCELL Technologies). Clinical data were determined by the UAB clinical lab- transcripts, termed the type I IFN signature (1, 2). Previous oratory and attending physician. All flow cytometry and bulk cell gene studies have identified unique type I IFN signatures among expression data were collected in a double-blinded manner. *Division of Clinical Immunology and Rheumatology, School of Medicine, University (to I.S.); and NIH Grants P30-AR-048311 and P30-AI-027767 to support flow of Alabama at Birmingham, Birmingham, AL 35294; †Department of Cell, Develop- cytometry, single cell gene expression, and imaging analyses. mental, and Integrative Biology, University of Alabama at Birmingham, Birmingham, ‡ Address correspondence and reprints requests to Dr. John D. Mountz, Division of AL 35294; Division of Rheumatology, Lowance Center for Human Immunology, x Clinical Immunology and Rheumatology, Department of Medicine, University of Emory University School of Medicine, Atlanta, GA 30322; and Birmingham VA Alabama at Birmingham, Shelby Interdisciplinary Biomedical Research Building, Room Medical Center, Birmingham, AL 35233 307, 1825 University Boulevard, Birmingham, AL 35294-2182. E-mail address: ORCID: 0000-0002-5119-7750 (A.L.M.). [email protected] Received for publication June 11, 2018. Accepted for publication August 14, 2018. The online version of this article contains supplemental material. This work was supported by National Institutes of Health (NIH) Grants R01-AI071110 Abbreviations used in this article: AA, African American; HC, healthy control; IFNP, and R01 AI134023, U.S. Department of Veterans Affairs/Biomedical Laboratory Re- IFN-producing signature; IFNP/R, mixed IFN and ISG producer/responder signa- search and Development Grants I01 BX004049 and I01 BX000600, and a Lupus ture; IFNR, IFN-responder signature; ISG, type I IFN–stimulated gene; MFI, mean Research Alliance (LRA) Distinguished Innovator Award (to J.D.M.); NIH Grant fluorescence intensity; SLE, systemic lupus erythematosus; UAB, University of R01-AI-083705 and a LRA Novel Research Award (to H.-C.H.); NIH Immunology Alabama at Birmingham. T32 Training Grant 2T32AI007051-39 and a Lupus Foundation of America Gina M. Finzi Memorial Student Summer Fellowship (to J.A.H.); NIH 5R37AI049660 Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 (to I.S.); Autoimmunity Center of Excellence – Emory University U19 AI110483 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800791 2 CUTTING EDGE: B CELL IFN-b IN SLE Study approval Results and Discussion These studies were conducted in compliance with the Helsinki Declaration and Type I IFN–producing and response genes in SLE Tr B cells approved by the institutional review board at UAB. All participants provided informed consent. We previously showed that type I IFN expression is a prominent feature of transitional stage 1 B cell development Flow cytometry analysis in BXD2 autoimmune mice and that T1 B cell IFN-b acts in Human Abs included BioLegend BV510–anti-CD24 (ML5), PE–anti-CD303 an autocrine priming mechanism to promote Ifna and ISGs (clone 201A), BV510–anti-IgM (clone MHM-88), Pacific Blue–anti-CD4 expression (22). Analysis of type I IFN gene expression from (clone RPA-T4), PE-Cy7–anti-CD10 (clone HI10a), BV650–anti-CD27 SLE patients revealed a significant increase in the expression (O323), Pacific Blue–anti-CD19 (HIB19), PE-Cy7–anti-CD38 (HB-7); SouthernBiotech PE-IgD (IADB6); and PBL Assay Science FITC–anti– of IFNB, IFNA1, IFNA14, IFNA17, and MX1 in Tr B cells IFN-b (MMHB-3). Dead cells were excluded from analysis with Fixable from AA patients with SLE (Supplemental Fig. 1A). To de- Viability Dye eFluor 780 (eBioscience). For intracellular staining, cells were termine whether distinct type I IFN and type I ISG gene stained with eFluor 780 viability dye, followed by fixation in 2% PFA and expression patterns were present in SLE B cells, Tr B cells 70% ice-cold methanol permeabilization prior to staining. Purity was vali- dated by postsort analysis of FACS-sorted cells to verify that .99% of cells from three female AA SLE subjects as described in fell into the sort gate after resorting. FACS data were acquired with an LSR II Supplemental Fig. 1B were FACS sorted and analyzed for FACS analyzer (BD Biosciences) and analyzed with FlowJo software (Tree expression of IFNB, IFNA, and ISGs (Fig. 1A). Hierarchical Star, Ashland, OR). clustering analysis revealed three prominent clusters with Superresolution structured illumination microscopy distinct gene signatures, including a mixed IFN and ISG Negative selection–purified B cells (STEMCELL Technologies) were fixed in producer/responder signature (IFNP/R), an IFN-responder Downloaded from 2% paraformaldehyde and permeabilized with Triton X-100, followed by signature (IFNR), and an IFN-producing signature (IFNP) blocking with 2% BSA. Cells were stained with AF647 goat anti-human IgM (Fig. 1A, 1B, Supplemental Table I). Cells from all three b (SouthernBiotech), FITC mouse anti-human IFN- (clone MMHB-3; PBL patients were equally represented in each cluster, revealing the Assay Science), or polyclonal rabbit anti-human IFN-b (Abcam), followed by anti-rabbit IgG AF488 secondary Ab.