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αProducing IFN-in Late-Stage Lupus Mice Defective in Cutting Edge: Plasmacytoid Dendritic Cells

LuoKaryala, Christopher M. Reilly, S. Ansar Ahmed and Xin M.Jingjing Ren, Alec M. Reihl, Husen Zhang, Saikumar V. Xiaofeng Liao, Song Li, Robert E. Settlage, Sha Sun,

ol.1501157http://www.jimmunol.org/content/early/2015/10/06/jimmun

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Cutting Edge: Plasmacytoid Dendritic Cells in Late-StageLupus Mice Defective in Producing IFN-aXiaofeng Liao,*,1 Song Li,†,1 Robert E. Settlage,‡,1 Sha Sun,x Jingjing Ren,*Alec M. Reihl,* Husen Zhang,{ Saikumar V. Karyala,‡ Christopher M. Reilly,‖

S. Ansar Ahmed,* and Xin M. Luo*

Plasmacytoid dendritic cells (pDCs) are professionaltype I IFN producers believed to promote lupus. How-ever, questions exist about whether they function at thesame level throughout the course of lupus disease. Weanalyzed high-purity pDCs sorted from lupus mice.Although pDCs produced a large amount of IFN-aduring disease initiation, those sorted from late-stagelupus mice were found to be defective in producingIFN-a. These pDCs expressed an increased level ofMHC, suggesting a functional drift to Ag presentation.We examined the potential mechanism behind the de-fect and identified a novel transcriptional factor, Foxj2,which repressed the expression of several genes inpDCs, but not IFN-a. Dysregulation in pDCs appearsto be predisposed, because they exhibited an alteredtranscriptional profile before the onset of clinical signs.Our results suggest that pDCs do not function thesame throughout the disease course and lose the abilityto produce IFN-a in late-stage lupus mice. TheJournal of Immunology, 2015, 195: 000–000.

It has been long recognized that type I IFNs, includingIFN-a, facilitate the progression of systemic lupuserythematosus (SLE). Based on large amounts of sup-

porting evidence obtained using human patient samples (1–6)and murine models (7–11), four Abs targeting IFN-a or typeI IFNR have been tested in human clinical trials for SLE (12–16). A couple of them showed promising efficacy (13–15). Abeneficial effect was observed in SLE patients with a low typeI IFN signature (i.e., those with lower expression of IFN-responsive genes); IFN-a blockade partially inhibited suchexpression. This suggests that early intervention (when the

IFN signature is still low) may be required for IFN-a–targeted therapies for SLE. Interestingly, IFN signaturesreturned to pretreatment levels in all patients by 6 mo afterthe last dose of anti–IFN-a Abs (17), suggesting that type IIFNs are constantly produced and pathogenic throughoutdisease progression. Because virtually all cells can produceIFN-a upon stimulation, identifying which types of cells playa predominant role in driving early- and late-stage SLE hasbecome an interesting, but challenging, question.Plasmacytoid dendritic cells (pDCs) were identified as

professional IFN-producing cells (18, 19). They producea large amount of IFN-a in response to TLR7/9 ligation (20).Studies of SLE patient samples revealed that immune com-plexes in the patient sera are also capable of inducing IFN-ain pDCs (21, 22), a process in which neutrophil extracel-lular traps are involved (23, 24). Recently, two groups ofresearchers independently showed that depletion of pDCsfrom mouse models of SLE ameliorates lupus-like disease (25,26). Deletion of pDCs in predisease lupus-prone mice sig-nificantly reduced lymphadenopathy and improved kidneypathology later in life. Strikingly, the benefit was sustainedeven after the pDC population recovered (26). This suggeststhat pDCs contribute to disease early in SLE pathogenesis andthat the recovered pDCs present in late-stage lupus micemight be less pathogenic.In this study, we used a classical mouse model of SLE,

MRL/Mp-Faslpr (lpr), as well as its parent strain, MRL, toexplore pDC functions in lupus mice. Female lpr mice ex-hibit systemic autoimmunity and glomerulonephritis anddie at an average age of 18 wk. Like SLE patients (27),diseased lpr mice show an elevated level of IFN-a in thecirculation (10, 28), but the source of this cytokine remainsunclear.

*Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College ofVeterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg,VA 24061; †Department of Crop and Soil Environmental Sciences, College of Agricul-ture and Life Sciences, Virginia Polytechnic Institute and State University, Blacksburg,VA 24061; ‡Virginia Bioinformatics Institute, Blacksburg, VA 24061; xDepartment ofDevelopmental and Cell Biology, University of California, Irvine, Irvine, CA 92697;{Department of Civil and Environmental Engineering, College of Engineering, VirginiaPolytechnic Institute and State University, Blacksburg, VA 24061; and ‖Edward ViaCollege of Osteopathic Medicine, Blacksburg, VA 24060

1X.L., S.L., and R.E.S. contributed equally to this work.

ORCIDs: 0000-0002-8133-3944 (S.L.); 0000-0002-0044-6403 (S.S.).

Received for publication May 22, 2015. Accepted for publication September 16, 2015.

This work was supported by X.M.L.’s startup funds, as well as by the Virginia Bioin-formatics Institute and Fralin Life Science Institute Small Grants Program (VBI/Fralin-GRL-01).

The RNA sequence data presented in this article have been submitted to BioProjectunder accession number PRJNA284002.

Address correspondence and reprint requests to Dr. Xin M. Luo, Department of Bio-medical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine,Virginia Polytechnic Institute and State University, 295 Duck Pond Drive, Blacksburg,VA 24061. E-mail address: xinluo@vt.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: MHC-II, MHC class II; pDC, plasmacytoid dendriticcell; RNA-seq, RNA sequencing; siRNA, small interfering RNA; SLE, systemic lupuserythematosus.

Copyright� 2015 by TheAmerican Association of Immunologists, Inc. 0022-1767/15/$25.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501157

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ImmunologyCutting Edge

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Materials and MethodsMice and cell sorting

Mice were purchased from The Jackson Laboratory and bred in-house. Allmice used were female. pDCs were sorted as CD11c+CD11b2PDCA1+B220+.Siglec-H inhibits IFN-a induction and was not used for sorting. Anti-mouseAbs were from eBioscience. RNA sequencing (RNA-seq) was performed, andthe data can be found under BioProject accession PRJNA284002.

Cell culture and analyses

CpG (ODN1585, 5 mM) was used to stimulate sorted pDCs or total bonemarrow cells. For gene knockdown, mouse Foxj2 small interfering RNA(siRNA) and negative control were purchased from QIAGEN. For quanti-tative PCR of cDNA, iTaq Universal SYBR Green Supermix and ABI 7500Fast System were used, with ribosomal protein L32 as the housekeeping gene.For FACS, cells were blocked with Fc, stained, and analyzed with a BDFACSAria II. Mouse IFN-a levels were measured with a PBL ELISA kit.

Statistical analysis

Analysis of nonsequencing data was performed with the Student t test, one-way ANOVA, and the Tukey posttest. The results were considered statisticallysignificant when p , 0.05. Error bars denote SEs.

Results and DiscussionIFN signatures in lupus pDCs

Early depletion of pDCs from lupus mice ameliorates disease(26). However, whether pDCs are the primary source of IFN-afor both early- and late-stage lupus remains unclear. We sortedpDCs (Fig. 1A) from the bone marrow of 6- and 16-wk-oldfemale lpr mice, representing predisease and late-stagelupus disease, respectively, and analyzed the gene expressionprofile. As controls, pDCs were sorted from the bone marrowof age-matched female MRL mice. MRL mice carry wild-typeFas and display lupus much later than do lpr mice. Theyappear normal at 6 wk and are considered to be at prediseasestage at 16 wk (29). Therefore, our mouse groups included16-wk-old lpr (late-stage lupus), 6 wk-old lpr and 16-wk-oldMRL (predisease), and 6-wk-old MRL mice (control). Al-

though mouse pDCs are most enriched in the bone marrowcompared with other organs, they are still a minor populationin the bone marrow (29). Rigorous quality control measureswere taken to ensure accurate processing of samples. Mea-surement of pDC-specific transcription factor E2-2 showedhigh purity of our sorted cells (Fig. 1B). Because B cell pro-genitors in the bone marrow share surface markers withpDCs, we measured the expression levels of B cell genesCd20, Ebf1, Vpreb3, and Igha. Less than 5% of our sortedpDCs might have been contaminated with B cell progenitors(data not shown). With high-purity pDCs, we found thatmany IFN-responsive genes were upregulated when com-paring 16 wk-old lpr mice with age-matched MRL mice(Fig. 1C, highlighted with yellow bars). This suggests thatbone marrow pDCs in late-stage lpr mice exhibit both type Iand II IFN signatures, indicating previous exposure to IFNs.

pDCs from late-disease lupus mice are unable to produce IFN-a uponCpG stimulation

We next identified signaling pathways based on the down-stream transcript signatures. Among five top canonical path-ways identified, four were found in the comparison between16-wk-old lpr mice and age-matched MRL mice (Fig. 2A). Acommon feature of the four pathways was upregulation ofMHC molecules (data not shown). Detection of MHC classII (MHC-II) on the surface of pDCs confirmed its upregu-lation in lpr mice with late-stage disease (Fig. 2B). Thissuggests that pDCs from late-stage lupus mice might excel atAg presentation, a phenomenon similar to human SLE inwhich patient pDCs were shown to be better APCs thannormal pDCs (30).We next questioned whether pDCs isolated from older lpr

mice, expressing a higher level of MHC-II, were still capableof producing IFN-a upon stimulation. A previous studyshowed some defect in IFN-a production from a mixedpopulation of bone marrow dendritic cells from late-stagelupus mice (31). With sorted pDCs of high purity, weshowed almost complete loss of CpG-mediated induction ofIFN-a from late-stage lupus pDCs (Fig. 2C). Interestingly,pDCs from 16-wk-old MRL mice (predisease) showed en-hanced IFN-a production, consistent with a significantlylower level of MHC-II on the surface of these cells (Fig. 2B).This suggests that pDCs produce a large amount of IFN-ato initiate the disease. Although the bone marrow has thehighest percentage of pDCs (29), lpr mice with late-stage dis-ease possess very large lymph nodes, increasing the absolutenumbers of pDCs. Indeed, we found that pDCs were pri-marily found in lymph nodes in 16-wk-old lpr mice (Fig. 2D,Supplemental Fig. 1A). However, lymph node pDCs werealso found to be defective in producing IFN-a (Fig. 2E). Thissuggests that the high level of IFN-a in late-stage lpr mice(10, 28) may not have come from pDCs. Additionally, wemeasured the transcript level of Atg7, a gene required forIFN-a induction by immune complexes (32) and found it besignificantly lower in pDCs from 16-wk-old lpr mice com-pared with age-matched MRL mice (Fig. 2F). This suggeststhat immune complex–mediated IFN-a production frompDCs may be defective as well. Interestingly, we found a sig-nificantly higher level of Sca1 in late-stage lupus pDCs; incontrast, the expression of this gene was negligible in pDCsfrom 16-wk-old MRL mice (Fig. 2G). This observation is

FIGURE 1. IFN signatures in pDCs sorted from lupus mice. (A) Sorting

strategy. (B) Transcript level of pDC-specific gene E2-2 in sorted pDCs

compared with pDC-depleted bone marrow cells (other cells). (C) Heat maps

of types I and II IFN-responsive genes (IRGs). Red, upregulation. Green,

downregulation. Yellow bars highlight the genes that were upregulated in

older lpr mice versus older MRL mice.

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consistent with a previous report that Sca1 expression onpDCs correlated negatively with their ability to produceIFN-a (33). Lastly, we confirmed that the defective abilityof pDCs to produce IFN-a in late-stage lupus mice was notrestricted to the lpr model. In another classical SLE mousemodel, NZB/W F1 pDCs exhibited a reduced ability toproduce IFN-a during late-stage disease compared with earlydisease (Fig. 2H). These results suggest that pDCs from late-disease lupus mice lose their ability to produce IFN-a. In-stead, they express more MHC molecules and may becomebetter APCs.

Defect in IFN-a production in pDCs is independent of thetranscriptional repressor Foxj2

We next investigated the mechanism behind the functional lossof pDCs to produce IFN-a. Upstream receptors leading toIFN-a induction in pDCs, including TLR9 for DNA Agsand FcRs for immune complexes, were expressed in pDCsisolated from late-stage lupus mice (Fig. 3A, SupplementalFig. 1B). We also measured the transcript level of anotherpotential sensor, TLR7, and found it to be overexpressed inthese pDCs (Supplemental Fig. 1C). This suggests that thedefect in IFN-a production was not due to a lack of upstream

FIGURE 2. pDCs from late-stage lupus mice unable to produce IFN-a upon CpG stimulation. (A) Ingenuity pathway analysis showing the top canonical

pathways. (B) MHC-II expression on bone marrow pDCs. (C) Production of IFN-a in sorted bone marrow pDCs upon CpG stimulation for 6 h. (D) Dis-

tribution of pDCs among bone marrow (black), spleen (gray), and mesenteric lymph node (white). (E) Production of IFN-a from sorted pDCs upon CpG

stimulation for 6 h. (F) Transcript level of Atg7 in sorted pDCs. (G) Transcript level of Sca1 in sorted pDCs. (H) Production of IFN-a from bone marrow pDCs

from 23-, 28-, and 33-wk-old NZB/W F1 female mice upon CpG stimulation for 6 h. *p , 0.05, **p , 0.01, ***p , 0.001, ANOVA, n $ 3 mice/group. BM,

bone marrow; C, older versus younger MRL; L, older versus younger lpr; MFI, mean fluorescence intensity; MLN, mesenteric lymph node; o, older; O, older lprversus older MRL; y, younger; Y, younger lpr versus younger MRL.

FIGURE 3. Defect in IFN-a production in pDCs independent of the transcriptional repressor Foxj2. (A) Transcript level of Tlr9 in sorted pDCs. (B)

Transcript level of IFNa in pDCs from older mice stimulated with CpG for 6 h. (C) Differentially expressed genes following the same or opposite pattern of

CpG-induced IFN-a. (D) Transcript level of Foxj2 in sorted pDCs and pDC-depleted bone marrow cells (Others). (E) Transcript levels of genes identified in (C)

in bone marrow cells of 16-wk-old lpr mice after transfection with negative-control siRNA (si-NC) or Foxj2-specific siRNAs (si-Foxj2). Fold differences are

shown. (F) Production of IFN-a in bone marrow cells (BMMC) of 4-mo-old lpr or MRL mice, transfected with siRNAs and stimulated with CpG for 6 or 18 h.

*p , 0.05, **p , 0.01, Student t test (n $ 3 mice per group). Log2FC, log2-fold changes.

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receptors. However, the problem seemed to be related totranscription or RNA stability, because pDCs from late-stagelupus mice expressed little IFN-a mRNA upon CpG stimu-lation (Fig. 3B). Thus, we explored a novel strategy of usingRNA-seq data to identify differentially expressed genes thatfollowed the same or opposite pattern of CpG-inducedIFN-a. We reasoned that common machineries might mod-ulate IFN-a transcripts together with other gene transcripts. Bystudying regulatory elements of the other genes, we might beable to identify the mechanism behind the defect in IFN-aproduction. To identify genes with the same expression pat-tern as CpG-induced IFN-a (Fig. 2C), we used the followingcriteria: expression in pDCs of older MRL mice had to be$2-fold higher than that of younger MRL mice, and ex-pression in pDCs of older lpr mice had to be $2-fold lowerthan that of older MRL mice. Similar criteria were applied toidentify genes with the opposite pattern. Using this strategy,we identified seven genes (Fig. 3C). We searched for sharedmicroRNAs using DIANA-microT but found no matches.However, promoter analysis revealed two shared cis-regulatoryelements that were present in the promoter regions of allgenes except one (Supplemental Fig. 1D). One of the twomotifs, TGTTT, can be bound by three members of theFox family of transcription factors (Supplemental Fig. 1E).Among them, only Foxj2 was expressed in pDCs. In lpr mice,Foxj2 was expressed significantly higher in pDCs than inother bone marrow cells (Fig. 3D). The other shared cis-regulatory element was an Rreb1 binding site, but Rreb1 wasnot preferentially expressed in pDCs (data not shown).We hypothesized that Foxj2 overexpression in pDCs re-

pressed the transcription of both IFN-a and genes with thesame pattern (Afap1, Numb, Tmem201). To test this hy-pothesis, we used siRNA to knock down Foxj2 in pDCs fromlate-stage lupus mice (Supplemental Fig. 1F). Such knock-down increased the transcript levels of Afap1, Numb, andTmem201 (Fig. 3E). Although little is known about thefunctions of these three genes in pDCs, our results suggestthat Foxj2 is a functional transcriptional repressor involved inshaping the gene expression profile of pDCs in late-stage lu-pus mice. Unexpectedly, however, knocking down Foxj2 didnot restore IFN-a production in pDCs from lpr mice (Fig.3F). In fact, Foxj2 seems to be required for the production ofIFN-a in pDCs, because Foxj2-specific siRNA decreasedIFN-a levels for both lpr and MRL mice. In addition, siRNAof Foxj2 did not change the expression of Atg7 (SupplementalFig. 1G), a gene required for IFN-a induction (32). Theseresults suggest that the defect in IFN-a production in pDCsof late-stage lupus mice is independent of Foxj2. Other po-tential mechanisms that we will explore in the future to tryto explain the defect include the involvement of lysosome-related organelle complexes, such as BLOC1 (Supplemental Fig.1H), whose downregulation might hinder TLR7/9-inducedIFN-a production in pDCs (34); a possible role for apoptosis,because pDCs from late-stage lupus mice appeared to expressa decreased level of the antiapoptotic gene Bcl-xl; and up-regulation in older lpr mice of Ptpn21 (also called PTPD1),a tyrosine phosphatase that interacts with Tec family kinasesto increase the activation of Stat3 (35), and Stat3 can nega-tively regulate the type I IFN response (36).Additional comparisons revealed that only 25 genes were

differentially expressed when comparing pDCs of older versus

younger lpr mice (Supplemental Fig. 2A, blue, false discoveryrate , 0.1). Among these genes, only nine genes reacheda p value of 0.05 (Supplemental Fig. 2B). Many of these areIFN-responsive genes, such as Ptpn11 and Pde5a, which wereupregulated in pDCs of early diseased mice (Fig. 1C) butsignificantly downregulated in pDCs of older lpr mice. This isconsistent with the observation that pDCs from late-stagelupus mice produced less IFN-a. In contrast to the identifi-cation of very few gene differences between pDCs of olderversus younger lpr mice, 207 genes were differentiallyexpressed when pDCs in older versus younger MRL micewere compared (Supplemental Fig. 2A, green). This suggeststhat there might be more changes in transcript abundance inpDCs prior to the onset of disease, whereas few genes changeas lupus progresses from the predisease stage to the late stage.Thus, we hypothesized that an altered gene expression profile(i.e., a lupus-prone profile) might already be present in pDCsprior to disease onset. To identify lupus-prone genes inde-pendent of Faslpr mutation, because few SLE patients carrymutations of the Fas gene, we investigated whether pDCsfrom older MRL mice at the predisease stage of lupus shareddifferentially expressed genes with pDCs from younger lprmice, which represents the predisease stage for the lpr mousestrain. We identified 54 such genes, including 44 protein-coding genes (Supplemental Fig. 2C, 2D) and 10 long un-annotated transcripts (Supplemental Fig. 2E, 2F). The sig-nificance of the lupus-prone profile in pDCs remains to beexplored. However, our findings raise an important questionof whether pDCs in SLE patients or lupus mice are born withthe predisposed, dysregulated gene expression profile. In fu-ture investigations, we will study pDCs from even youngermice to determine potential early defects, as well as whethertriggers later in life might manifest the defects.

DisclosuresThe authors have no financial conflicts of interest.

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