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

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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 (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 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. DAPI nuclear stain was included for presence of these major clusters in different SLE patients nucleus determination (200 ng/ml). (Fig. 1C). Superresolution imaging was carried out using the Nikon N-SIM E Cells within the IFNP/R cluster expressed the highest levels http://www.jimmunol.org/ superresolution microscope (resolution capable of 120 nm x–y and 300 nm z). Images were acquired with a 1003 1.49 numerical aperture objective; of Tr B cell marker CD24 (Fig. 1D, upper left). Cells within ORCA-Flash 4.0 v2 scientific complementary metal oxide semiconductor the IFNP/R cluster also expressed higher levels of IFNB, camera (Hamamatsu); 488-, 561-, and 640-nm laser excitation; a mul- IFIT1, IRF7, IRF9, ZBP1, IFNA1, IFNA7, and CCND1 tipass dichroic cube; and specific emission filters. DAPI images were compared with the IFN or IFN cluster or both (Fig. 1D, acquired with a widefield excitation and are at a conventional resolution. R P Images were acquired and reconstructed with Nikon Elements software. Supplemental Table I). This is consistent with our previous Staining intensity and localization of IFN-b were carried out using observations in BXD2 mice in which IFNB expression was Fiji/ImageJ. upregulated in early T1 B cells (22). Cells within the IFNR cluster expressed higher levels of IFNAR1, MX1, IFIT2, PKR, Real-time quantitative RT-PCR by guest on October 4, 2021 RIG1, and CCND2 (Fig. 1E). The IFNP cluster was charac- RNA isolation, cDNA synthesis, and real-time PCR reactions were carried out terized by higher levels of IFNA4, IFNA5, IFNA10, IFNA14, as described previously (20, 21). Primers for GAPDH are as follows: forward, 59-GCACTCACTGGAATGACCTC-39; backward, 59-TTCTTCGCACT- IFNA16, and IFNA17 (Fig. 1F). Expression of TLR3 and GACACACTG-39. All primers used for single-cell analysis are described in TLR7 but not TLR9 was different among the groups Supplemental Table I. (Supplemental Fig. 1C). Single-cell gene expression and cell Single-cell gene expression analyses identities were validated by analysis of CD20, CD3, and CD303 expression (Supplemental Fig. 1D). Together, the results Gene expression analysis of single transitional (Tr) B cells (CD24+CD38+ 2 IgD+CD27 ) obtained from SLE PBMCs was performed using the Flu- reveal heterogeneous type I IFN–producing and –responding idigm single-cell capture and BioMark RT-PCR analysis system (Fluidigm, signatures in circulating Tr B cells, suggesting that B cells are South San Francisco, CA) as described in detail previously (22). Single-cell not only type I IFN targets but also producers of type I IFNs gene expression clustering analysis was carried out using the ClustVis online in SLE. web tool (23). Single-cell gene expression data can be retrieved at https:// biit.cs.ut.ee/clustvis/?s=zlbtSCZKfAcPrUg. Increased intracellular IFN-b in SLE B cells In vitro B cell secretion of type I IFN assay FACS staining (Fig. 2A) and confocal imaging (Fig. 2B) of b b B cell production of functional type I IFNs was analyzed using coculture IFN- revealed increased levels of IFN- in B cells from SLE of purified primary B cells (5.0 3 105 cells per well) with HEK-Blue patients compared with healthy controls (HCs). Quantifica- IFN-a/b cells that expressed an inducible secreted embryonic alkaline tion of IFN-b mean fluorescence intensity (MFI) revealed phosphatase (SEAP) (InvivoGen, SanDiego,CA).Bcellspurifiedusing that although Tr and naive B cells from SLE patients the Human Pan B Cell Isolation Kit (Miltenyi Biotec) were unstimu- b latedorstimulatedfor10minwithanF(ab’)2anti-hIg(IgM+IgG)Ab exhibited significantly increased IFN- compared with HCs, (Thermo Fisher Scientific) (10 mg/ml) plus 5 mg/ml poly(I:C) or in CD4 T cells and CD303+CD4low plasmacytoid dendritic 5 mg/ml CL264 (InvivoGen) before HEK-Blue IFN-a/b cells (2.5 3 b 4 cells (24), IFN- levels were not significantly increased in SLE 10 cells per well) were added into the medium. Supernatants were b collectedatthe24-htimepointandwereincubatedfor1hwiththe compared with HCs (Fig. 2A). Staining of IFN- was spe- QUANTI-Bluecolorimetricenzymeassayreagentfordeterminationof cifically inhibited by preincubation with human IFN-b but absorbance at OD650. not mouse IFN-a (Supplemental Fig. 2A). We next deter- b Statistics mined the subcellular localization of IFN- in SLE B cells. Two anti–IFN-b Abs detected a mainly cytoplasmic distri- 6 6 Results are mean SD or mean SEM as described in the figure leg- bution of IFN-b in IgM+ B cells (Fig. 2C, Supplemental ends. The p values ,0.05 were considered significant. Unless otherwise indicated, all analyses were performed using GraphPad Prism software Fig. 2B). Although significantly less prominent compared (La Jolla, CA). with the cytoplasmic localization, IFN-b was also detected in The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on October 4, 2021

2 FIGURE 1. Type I IFN and ISG gene expression in single Tr B cells from SLE patients. Tr B cells (CD24+CD38+IgD+CD27 ) isolated from PBMCs of three SLE patients (see Supplemental Fig. 1B) were prepared for single-cell gene expression analysis. (A) Heat map of hierarchically clustered type I IFN gene expression clustering in individual B cells (n = 207). The top row above the heat map is color coded to denote cell clustering and SLE patient origin. (B) Principal component (PC) analysis of SLE Tr B cell clusters. The x- and y-axis show PC1 and PC2 that explain 15.9 and 9.3% of the total variance, respectively. PC analysis was carried out based on PC1 and PC2 segregation of 32 genes in the IFNP/R, IFNP, or IFNR B cell clusters. Prediction ellipses are such that with p = 0.95, a new observation from the same group will fall inside the ellipse. (C) Bar graph showing the number of single cells designated to each type I IFN cluster based on the gene expression profile from each individual SLE patient (x2 analysis). (D–F) Dot plots showing the normalized expression of representative genes in D E F 6 the IFNP/R cluster ( ), the IFNR cluster ( ), and the IFNP cluster ( ) of B cells as defined by hierarchical clustering. All results are mean SD. Significant differences among means were analyzed using a one-way ANOVA test with the p value shown on the top of each graph. Differences between groups were analyzed using Tukey multiple comparisons test. Results are shown as mean 6 SD. *p , 0.05, **p , 0.01, *** p , 0.005 between the indicated groups. the nucleus (Fig. 2C) as previously reported for other cell monomer and dimer (27, 28). Together, these results confirm types (25, 26). Western blot analysis of cytoplasmic extracts the presence of cytoplasmic IFN-b in SLE B cells. from isolated SLE B cells ex vivo further confirmed the To determine if B cells produced biologically active type I presence of a 25- and 50-kDa band (Supplemental Fig. 2C) IFNs, an HEK IFN-a/b reporter cell line assay was carried consistent with the predicted molecular mass of human IFN-b out. Stimulation of the human B cell lymphoma cell line 4 CUTTING EDGE: B CELL IFN-b IN SLE Downloaded from http://www.jimmunol.org/

FIGURE 2. Increased expression of intracellular IFN-b in B cells from a subset of SLE patients. (A) Gating strategy (upper left), representative histograms (lower left), and summary of IFN-b MFI (right) in circulating Tr B cells, naive B cells, CD4 T cells, and plasmacytoid dendritic cells (pDCs) in SLE compared with HC. (B) Confocal microscopy imaging (left) and bar graph quantitation of IFN-b intensity (right) in purified B cells from a representative HC or an SLE patient (objective lens 320). (C) Structured illumination microscopy superresolution imaging and analysis of IFN-b intracellular localization in representative B cells from SLE patients. Top, Representative images showing intra- versus extranuclear staining of IFN-b. The nucleus–cytoplasmic border was defined by DAPI staining (dotted white line) (objective lens 3100). Bottom, ImageJ quantitation of intra- and extranuclear intensity (left) and distribution (right) of IFN-b (n = 22 cells from three SLE patients). (D) HEK-Blue reporter cell analysis of type I IFN secretion by B cells from SLE (n =9)orHC(n = 3) under the indicated conditions of stimulation. (E) Correlation of FACS detection of baseline (ex vivo) B cell IFN-b (MFI) with HEK-Blue analysis of IFN-b secretion from anti-Ig by guest on October 4, 2021 plus poly(I:C) stimulated B cells. All results are shown as mean 6 SD (unpaired Student t test or Mann–Whitney U test for nonnormally distributed data). *p , 0.05, **p , 0.01, ***p , 0.005.

Ramos with TLR3 ligand poly(I:C) induced the highest (Fig. 3A). Subjects who were positive for anti-Sm at any time IFN-b response compared with a TLR7 ligand (CL264) and a during their disease course exhibited a significant increase in TLR9 ligand (ODN-2006) as measured by both intracellular IFN-b levels in Tr and CD27+ memory B cells, whereas FACS for IFN-b (Supplemental Fig. 2D, left) and by the subjects who were seropositive for anti-SSA exhibited in- HEK reporter assay (Supplemental Fig. 2D, right). Stimula- creased IFN-b expression in Tr, naive, and CD27+ B cells tion of purified SLE B cells with anti-Ig plus TLR3 induced (Fig. 3B, 3C). These data suggest an association between an increased HEK reporter response compared with B cells IFN-b expression and enhanced survival of B cells exhibiting derived from HCs (Fig. 2D). As an additional control, levels reactivity with nucleic acid/ complexes able to coac- of IFN-b measured in the FACS assay were correlated with tivate BCR and TLR signaling (31). IFN-b protein secretion as measured by the HEK IFN-a/b SLE subjects with a history of renal disease also exhibited a reporter assay (Fig. 2E). These results are consistent with significant increase in IFN-b in Tr and naive B cells compared previous findings by Gram et al. (29), which reported type I with SLE patients without a history of renal disease (Fig. 3D). IFN secretion by human B cells upon poly(I:C) stimulation and Interestingly, intracellular IFN-b in Tr and naive B cells was further suggest the importance of further evaluation of in vivo significantly higher in AA compared with non-AA patients TLR3 ligands, including U1 RNA that may be associated with (Fig. 3E). Levels of IFN-b in SLE B cells were generally B cell secretion of functional type I IFN in SLE patients (30). significantly higher compared with HCs, even in comparisons between autoantibody-negative or otherwise low-severity pa- Intracellular IFN-b is associated with autoantibody production, tients and HCs (Fig. 3A–E). B cell endogenous IFN-b levels renal disease, and AA race. The higher expression and produc- were not significantly different in SLE subjects with low tion of IFN-b from SLE B cells suggest that B cell intracellular complement or other clinical parameters, including the SLE IFN-b may be an important factor associated with SLE Disease Activity Index or hydroxychloroquine treatment, but pathogenesis. We identified that patients who were seroposi- were significantly higher compared with HCs (Fig. 3F–H). tive for anti-dsDNA at the time of specimen collection These results suggest that B cell IFN-b is most strongly exhibited significantly increased levels of IFN-b (MFI) in Tr associated with increased autoantibodies and renal disease and and CD27+ memory B cells compared with SLE patients who that polymorphisms in the IFN-b enhanceosome genes or were seronegative for anti-dsDNA at the time of collection other upstream genes may predispose some patients to the The Journal of Immunology 5

FIGURE 3. Elevated B cell IFN-b in autoantibody-positive AA SLE patients. The MFI of IFN-b in the indicated populations of B cells in HCs or in SLE Downloaded from patients, segregated by (A) positivity of anti-dsDNA, (B) historic positivity of anti-Sm or (C) anti-SSA, (D) the presence of renal disease, (E) race (non-AAs versus AAs), (F) complement (C3/C4) normal versus low, (G) SLE Disease Activity Index #4 versus .4, and (H) with or without hydroxychloroquine (HCL) treatment. All clinical characteristics, except anti-Sm and anti-SSA, were collected at the time of PBMC sample collection. Results are mean 6 SD. Statistical differences were determined by Mann–Whitney U test. *p , 0.05, **p , 0.01, ***p , 0.005 between the indicated comparisons. development of type I IFN dysregulation and autoimmune 5. Sharma, S., Z. Jin, E. Rosenzweig, S. Rao, K. Ko, and T. B. Niewold. 2015. Widely divergent transcriptional patterns between SLE patients of different ancestral http://www.jimmunol.org/ disease (32). This notion is supported by recent population- backgrounds in sorted immune cell populations. J. Autoimmun. 60: 51–58. level studies that identified the IFNB locusasatrans-regulatory 6. Rai, R., S. K. Chauhan, V. V. Singh, M. Rai, and G. Rai. 2016. RNA-seq analysis reveals unique transcriptome signatures in systemic lupus erythematosus patients hotspot that controlled antiviral networks enriched in genes with distinct autoantibody specificities. PLoS One 11: e0166312. differentially expressed in AA versus European American 7. El-Sherbiny, Y. M., A. Psarras, M. Y. M. Yusof, E. M. A. Hensor, R. Tooze, healthy volunteers (33). Together, the present findings sup- G. Doody, A. A. A. Mohamed, D. McGonagle, M. Wittmann, P. Emery, and E. M. Vital. 2018. 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