IFN Sulfate on B Cells Via the Action of Type I Virally-Induced

Virally-Induced Upregulation of Heparan Sulfate on B Cells via the Action of Type I IFN

This information is current as Nadine Jarousse, Damian L. Trujillo, Sarah of September 28, 2021. Wilcox-Adelman and Laurent Coscoy J Immunol 2011; 187:5540-5547; Prepublished online 2 November 2011; doi: 10.4049/jimmunol.1003495

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Supplementary http://www.jimmunol.org/content/suppl/2011/11/02/jimmunol.100349 Material 5.DC1 http://www.jimmunol.org/ References This article cites 35 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/187/11/5540.full#ref-list-1

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Virally-Induced Upregulation of Heparan Sulfate on B Cells via the Action of Type I IFN

Nadine Jarousse,*,1 Damian L. Trujillo,*,1 Sarah Wilcox-Adelman,† and Laurent Coscoy*

Cell surface heparan sulfate (HS) is an important coreceptor for many cytokines, chemokines, and growth factors. In this study, we report that splenic murine B cells express very little HS and that upon infection with either gammaherpesvirus (murine gammaherpesvirus 68) or betaherpesvirus (murine cytomegalovirus), HS is rapidly upregulated at the surface of B cells. HS upregulation was not observed in mice deficient for the type I IFN (IFN-I) receptor. Additionally, treatment of wild-type mice with the IFN-I inducer polyinosine polycytidylic acid triggered HS expression at the B cell surface. Similarly, incubation of purified splenic B cells with IFN-I, TLR ligands, or BCR stimulators ex vivo resulted in a drastic increase in HS surface expression. We found that IFN-I induced an increase in the surface expression of HS-modified syndecan 4 as well as that of an unidentified heparan sulfate pro-

teoglycan. Finally, IFN-I treatment increased B cell responsiveness to APRIL, a cytokine involved in B cell survival and T cell- Downloaded from independent B cell responses. Enzymatic removal of HS from IFN-I–treated B cells inhibited APRIL. Altogether, our results indicate that upon herpesvirus infection in mice, HS is rapidly upregulated at the surface of B cells due to the action of IFN-I, potentially increasing B cell responsiveness to cytokines. Induction of HS expression at the B cell surface by stimulators of the innate immune response likely plays a key role in the development of a robust immune response. The Journal of Immunology, 2011, 187: 5540–5547. http://www.jimmunol.org/ eparan sulfate (HS) is a glycosaminoglycan abundantly HS is commonly described as a ubiquitously expressed gly- expressed as proteoglycans on most cell surfaces and in cosaminoglycan, but to date its expression and function have been H the extracellular matrix (1). Upon synthesis in the Golgi examined mainly in adherent cells. Although many cytokines in- apparatus, HS is attached to a restricted group of core proteins volved in immune function are known to bind HS (IL-2 to IL-8, called heparan sulfate proteoglycans (HSPGs), consisting pri- IL-10, IL-12, IFN-g, TNF-a, MIP-1b, APRIL, etc.) (2), little is marily of members of the syndecan and glypican families (2). The known about the contribution of HS to lymphocyte biology. Al- study of various genetic mutants defective in HS biosynthesis has though HS molecules were shown to play a role in IL-7–depen- shown that HS plays an essential role in mammalian development dent B lymphopoiesis (8), the importance of HS chains at the and physiology (2). HS polysaccharides mediate interactions with surface of mature B cells is not clear. Intriguingly, HSPGs are by guest on September 28, 2021 a wide variety of extracellular ligands including growth factors, expressed at certain stages of B lymphocyte differentiation. For cytokines, chemokines, enzymes, and cell-adhesion molecules (2, example, syndecan 1 (CD138) is expressed on pre-B cells, absent 3). HS serves as a coreceptor in many cell signaling pathways and in circulating and peripheral B lymphocytes, and re-expressed modulates signal transduction (4). HS also facilitates cell adhesion upon differentiation to the plasma cell stage (9). Syndecan 4 is to the extracellular matrix and acts as a transporter of chemokines expressed on bone-marrow B cells and most mature B cell subsets across cells and as a stabilizer of morphogen gradients along (10). Although the expression of syndecans is tightly regulated, epithelial surfaces (2, 5, 6). HS “capture” of soluble protein their function in B cell biology are not defined. ligands from three-dimensional space converts them into a two- We recently reported that B cell lines express little to no HS at dimensional array that enhances further molecular encounters (7). their surface (11), which prompted us to examine the status of HS Thus, HS is involved in many physiological systems, orchestrating at the surface of naive splenic B cells. In this study, we report that cellular processes such as growth, division, adhesion, migration, naive splenic murine B cells express very little HS at their surface, and differentiation (1, 2). but that HS expression was upregulated upon a type I IFN (IFN-I) response in vivo. This upregulation was essential for the signaling of APRIL, an HS-binding cytokine (12) involved in class switch recombination and B cell survival (13, 14). Attachment of HS to *Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720; and †Boston Biomedical Research Institute, Boston, MA 02472 syndecan 4 and a 130-kDa HSPG accounts for the induction of HS 1N.J. and D.L.T. contributed equally to this work. on B cells. Altogether, our results suggest that in the context of an Received for publication October 22, 2010. Accepted for publication September 21, infection that triggers an IFN-I response, B cell responsiveness to 2011. cytokines and other HS-binding ligands might be amplified due to Address correspondence and reprint requests to Prof. Laurent Coscoy, Department of an upregulation of HS surface display. Molecular and Cell Biology, University of California, Berkeley, 142 Life Sciences, Addition #3200, Berkeley, CA 94720. E-mail address: [email protected] Materials and Methods The online version of this article contains supplemental material. Mice and viruses Abbreviations used in this article: HS, heparan sulfate; HSPG, heparan sulfate pro- C57BL/6 mice were obtained from The Jackson Laboratory. Mice deficient teoglycan; IFNAR, IFN-a receptor; IFN-I, type I IFN; KO, knockout; MCMV, murine cytomegalovirus; MHV68, murine gammaherpesvirus 68; poly I:C, polyinosine for the IFN-a receptor [IFNAR knockout (KO) mice] were a kind gift from polycytidylic acid; TACI, transmembrane activator and calcium modulator and cyclo- Dr. Daniel Portnoy, University of California, Berkeley. Bone marrow from philin interactor; wt, wild-type. syndecan 4-deficient mice was a kind gift from Dr. Sarah Wilcox- Adelman. Experiments were conducted with 6- to 12-wk-old mice in ac- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 cordance with institutional guidelines for animal care and use. Murine www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003495 The Journal of Immunology 5541 gammaherpesvirus 68 (MHV68) was obtained from the American Type 50UN, 107 cells/ml, 1 U/ml; chondroitinase: cat. no. C3667-10UN, 107 Culture Collection (VR-1465) and propagated on BHK-21 cells. Smith cells/ml, 0.005 U/ml; Sigma) for 3 h at 37˚C. Cells were then washed and strain murine cytomegalovirus (MCMV; MW97.01) was a kind gift from lysed prior to SDS-PAGE. For immunoprecipitation, lysate was precleared Dr. David Raulet, University of California, Berkeley. MCMV was propa- with protein A/G beads (cat. no. sc2003; Santa Cruz Biotechnology) and gated on NIH3T3 cells. Virus-containing supernatants were cleared of then immunoprecipitated using an anti-syndecan 4 Ab (cat. no. ab24511; cellular debris by low-speed centrifugation for 20 min. Virus was subse- Abcam). The immunoprecipitate was washed prior to SDS-PAGE. After quently concentrated by centrifugation at 24,000 3 g for 2 h and purified transferring to immobiline membrane (Thermo Fisher), the membrane was over a 30% sucrose cushion by centrifugation at 85,000 3 g. Pelleted virus probed first with anti-HS (clone 3G10, cat. no. 370260-1; Seikagaku) and was resuspended in PBS and stored at 280˚C. Viral titers were determined then with HRP-conjugated anti-mouse IgG. Western blots were developed on NIH3T3 cells using a standard plaque assay. BHK-21 and NIH3T3 cells using Super Signal West Pico Chemiluminescent Substrate (cat. no. 34080; were grown in DMEM supplemented with 10% FBS and 100 units/ml Pierce Biotechnology), and exposed onto film. penicillin and 100 mg/ml streptomycin. Statistical analysis Isolation and culture of splenic B cells All statistical results are expressed as mean 6 SEM. Statistical analysis was Splenocytes were isolated from C57BL/6 or IFNAR KO mice, and B cells performed using a nonparametric Mann–Whitney U test for comparison were purified using the EasySep negative selection mouse B cell enrichment between two groups (GraphPad software). Differences were considered kit (Stem Cell Technologies). B cell purity was assessed by flow cytometry significant at p , 0.05. using anti-CD19 or anti-B220 Abs (.98% purity). Cell viability was verified by propidium iodide or 7-aminoactinomycin D staining (.99% Results viability). Cells were resuspended at a density of 2 3 106 cells/ml (except for APRIL assay; see below) in RPMI media containing 100 mM MEM Naive B cells express low levels of surface HS nonessential amino acids, 55 mM 2-mercaptoethanol, 1 mM sodium py- To determine the expression level of HS on naive murine B cells, Downloaded from ruvate, and 10 mM HEPES. When indicated, reagents were added to the media: IFN-b at 1000 U/ml unless indicated otherwise (PBL IFN source), splenocytes from C57BL/6 mice were isolated, stained with a anti-IgM at 2 mg/ml (Jackson ImmunoResearch), type A and type B CpG commonly used anti-HS Ab (F58-10E4; Seikagaku), and analyzed oligonucleotides at 3 mM, and LPS at 1 mg/ml. by flow cytometry. B cells were identified using an anti-B220 Ab Flow cytometry (similar results were obtained using an anti-CD19 Ab). The murine NIH3T3 fibroblast cell line was used as a positive control (Fig. 1). Splenocytes or purified splenic B cells were washed in PBS with 1% BSA Similar to what we observed in B cell lines (11), we found that the and incubated with 1 mg/ml purified rat anti-mouse CD16/CD32 (Fc block; http://www.jimmunol.org/ BD Pharmingen) at 4˚C for 15 min, followed with an anti-HS Ab used at level of HS expression on the surface of splenic B cells is very low 1:100 (10E4 epitope, F58-10E4; Seikagaku) for 30 min at 4˚C. The isotype or undetectable. Likewise, van der Voort et al. (15) reported that control was a mouse IgM k (TEPC 183; Sigma). After two washes in PBS HS was hardly detectable at the surface of resting human tonsillar with 1% BSA, bound Ab was revealed by staining with an FITC- or PE- a B cells. Thus, it appears that neither murine nor human naive conjugated anti-mouse IgM (Igh-6a) mAb (BD Pharmingen). When B cells express a significant level of HS at their surface. staining for transmembrane activator and calcium modulator and cyclo- philin interactor (TACI), PE-conjugated anti-TACI (cat. no. 558410; BD HS is upregulated at the B cell surface after herpesvirus Pharmingen) was used. mAb KY/8.2 (cat. no. 550350; BD Pharmingen) infection in vivo was used to stain for syndecan 4. The isotype control for syndecan 4 was rat IgG2a (cat. no. MAB006; R&D Systems). When using total spleno- HS participates in cell surface binding of many cytokines involved by guest on September 28, 2021 cytes, cells were costained with an anti-B220 or anti-CD19 Ab directly in inflammation and also plays an important role in cell adhesion conjugated with a fluorochrome. When staining cultured B cells, dead cells were excluded from the analysis by propidium iodide or 7-amino- and migration (2). Notably, van der Voort et al. (15) showed that actinomycin D staining. HS was upregulated at the surface of human tonsillar B cells upon activation in culture. Thus, we speculated that HS expression Measure of APRIL-mediated IgA production might be upregulated at the surface of B cells during an immune 293T cells were transfected with either PCDNA3.1 (vector control) or response in vivo, and this upregulation might be important for a vector encoding soluble APRIL (a kind gift from Dr. Kimberley Fiona, B cell function. To test whether HS is upregulated on University of Amsterdam, Amsterdam, The Netherlands) using Lipofect- B lymphocytes in the context of an infection in vivo, wild-type amine 2000 (Invitrogen) according to the manufacturer’s instructions. Two days later, supernatant was harvested from transfected 293T cells and di- C57BL/6 mice were infected with MHV68 either by i.v. or i.p. luted 1:2 in RPMI that included supplements and FBS. Diluted supernatant infection. Mice were sacrificed at various time points postin- was used to resuspend purified splenic B cells (1 3 106 cells per ml). Then fection, and splenocytes were examined for HS expression as 2 3 105 cells/well (200 ml) were plated in a round-bottom 96-well plate. Cells were left untreated, treated with either IFN-b (45 U/ml) or with 3 units/ml of heparinase III (Sigma-Aldrich), or both simultaneously. Cells were cultured for 6 d. At days 2 and 4 postplating, an additional 0.6 U/well of heparinase was added to all heparinase-treated samples. At 6 d postplating, supernatant from wells was collected and added to ELISA plates coated with anti-IgA Ab (Southern Biotech). IgA production was measured with an alkaline phosphatase-conjugated anti-IgA Ab. Substrate conver- sion (pNpP, cat. no. N2770; Sigma) was monitored using a plate reader measuring at l 405 nm. A standard curve using known concentrations of IgA was generated and used to determine IgA concentration (ng/ml) in the B cell supernatants. Generation of bone marrow chimeras Female C57BL/6 congenic mice (CD45.1+) between 6 and 8 wk were lethally irradiated with 900 rad. Twenty-four hours later, the mice were injected (i.v.) with 5 3 106 bone marrow cells from syndecan-deficient FIGURE 1. Naive B cells express very little HS at their surface. Freshly mice (C57BL/6 background; CD45.2+). Ten weeks postinjection, recon- isolated murine splenocytes were stained with anti-HS Ab and an anti- stitution efficiency was checked by tail-vein bleeding. B220 Ab. The histogram shows the level of HS on B220-positive cells (left panel). NIH3T3 cells were tested for HS expression as a control (right Western blot and immunoprecipitation panel). Shaded histogram: isotype control. The y-axis is percent of max- Purified ex vivo B cells were treated or not with IFN-I, then were incubated imum. One representative experiment is shown from a total of more than with or without heparinase and chondroitinase (heparinase: cat. no. H8891- 10 repeats, where one to three mice were used per repeat. 5542 UPREGULATION OF HS ON B CELLS UPON VIRAL INFECTION described earlier. HS is upregulated at the B cell surface as early To test further the role of IFN-I in HS induction, we used another as 12 h after MHV68 i.v. infection (Fig. 2A, left panel). Impor- model of IFN-I induction in the absence of viral infection. Poly- tantly, HS upregulation is observed on the entire B cell population inosine polycytidylic acid (poly I:C) is a dsRNA mimetic that is (Fig. 2A, middle panel). The increase in HS surface expression is commonly used to trigger IFN-I production in vivo (19). Wild- also detected after i.p. infection (Fig. 2A, right panel). Although type (wt) C57BL/6 mice were treated with poly I:C (i.v. injection), HS induction on the entire B cell population as early as 12 h and HS expression was measured at the surface of splenic B cells. postinfection is unlikely to be the result of a direct interaction Enhanced HS expression was observed as early as 12 h after with MHV68, we verified that HS upregulation was also observed poly I:C injection (Fig. 3B, left panel). Notably, the induction of after infection with MCMV, a virus that does not target B cells. As HS after poly I:C injection was observed only on B cells, as the shown in Fig. 2B, MCMV infection also triggers an increase in non-B cell fraction of splenocytes showed no significant HS ex- HS expression at the B cell surface after either i.v. (Fig. 2B, left pression (Fig. 3B, right panel). panel) or i.p. (Fig. 2B, right panel) infection. In all cases, the To test the direct effect of IFN-I on B cells, in the absence of entire B cell population exhibits enhanced HS expression, sug- other cell types or cytokines, splenic B cells were purified from wt gesting the possible action of a diffusible agent such as a cyto- mice and cultured in the presence of recombinant IFN-b for 24 h. kine. Surface HS was examined by flow cytometry. Culturing purified B cells for 24 h, in the absence of IFN-I or any other stimulator, HS upregulation is mediated by IFN-I led to an upregulation of surface HS (Fig. 3C, media). Notably, IFN-I constitutes the first line of host defense against viruses (16). IFN treatment results in a dramatic increase in HS expression over

IFN-I plays a critical role in controlling both the acute and latent media alone (Fig. 3C, IFN-I). Importantly, the induction of HS on Downloaded from phases of MHV68 infection (17, 18). To test whether IFN-I is B cells is not due to HSPG binding TACI (Supplemental Fig. 1A), involved in HS upregulation in B cells after MHV68 infection, as IFN-I did not induce TACI on B cells. Altogether, our results IFNAR KO mice were infected with MHV68, and HS expression indicate that IFN-I–mediated signaling triggers upregulation of on B cells was examined at 36 h postinfection. Whereas HS was HS expression at the surface of B cells and accounts for the up- strongly induced on B cells from infected wild-type mice, the regulation observed in vivo upon MHV68 infection. Notably, the

level of HS expression on B cells from infected IFNAR KO mice increased HS on B cells upon culturing (Fig. 3C, media) is also http://www.jimmunol.org/ was close to background, comparable with that of control PBS- observed on B cells isolated from IFNAR KO mice (Fig. 3D, right injected mice (Fig. 3A), indicating a role for IFN-I in HS upreg- panel), indicating that signals other than IFN-I can trigger HS to ulation during an in vivo immune response. be upregulated in culture. by guest on September 28, 2021

FIGURE 2. HS is induced on the surface of splenic B cells after infection of mice with MHV68 or MCMV. A, C57BL/6 mice were injected with 5 3 105 PFU of MHV68 or PBS as a control. Mice were euthanized at different time points, and HS expression was analyzed on B cells as before. Left panel, Mice were injected i.v. Results are expressed as a fold induction over PBS-injected mice (PBS control set to 1). Middle panel, i.v., 24 h postinfection. Right panel, Mice were injected i.p. (48 h postinfection). B, C57BL/6 mice were injected i.v. (left panel) or i.p. (right panel) with 5 3 105 PFU of MCMV or PBS as a control, and HS expression on splenic B cells was analyzed as before after 24 h (i.v.) or 48 h (i.p.). The y-axis for all histograms is percent of maximum. The graph (A) and histograms (A, B) depict one representative experiment using 5–10 mice that was repeated at least three times. The Journal of Immunology 5543

FIGURE 3. IFN-I triggers HS upregulation at the B cell surface. A, C57BL/6 mice or IFNAR KO mice were injected i.v. with 5 3 105 PFU of MHV68 and examined as before for HS expression on B cells after 36 h. B, C57BL/6 mice were injected with 200 mg of poly I:C and analyzed as before for HS expression on B cells (CD19+) or non-B cells (CD192) 12 h later. C, B cells were puriﬁed from C57BL/6 mice and incubated for 24 h in media with or without IFN-b (1000 U/ml). HS expression was analyzed by ﬂow cytometry as before. D, Splenic B cells isolated from IFNAR KO mice were analyzed for HS expression either immediately after isolation (left panel) or after 24 h in culture (right

panel). In A–D, shaded histograms rep- Downloaded from resent an isotype control. The y-axis for all histograms is percent of maximum. All histograms are representative experiments using 3–10 mice that were repeated at least ﬁve times. http://www.jimmunol.org/

Activation of purified splenic B cells through the B cell Ag course of an infection, such as IFN-I, TLR ligands, or molecules receptor (via an anti-IgM Ab) also results in an increase in HS capable of cross-linking BCRs, cause HS to be upregulated in expression over media alone (compare Fig. 4A and 4B), consistent B cells. with an earlier study on human tonsillar B cells (15). Similarly, stimulation of TLR4 with LPS (Fig. 4C), TLR9 with type A (Fig. Syndecan 4 and a 130-kDa HSPG are modified with HS on 4D) or type B (Fig. 4E) CpG oligonucleotides leads to an up- B cells upon IFN-I treatment regulation of HS at the B cell surface. Although this upregulation We hypothesized that the increase in HS expression on B cells of HS is not dependent on IFN-I signaling, IFN-I does potentiate was due to an increase in expression of HSPGs. To investigate by guest on September 28, 2021 HS induction as demonstrated by the lower amount of HS ob- this possibility, we decided to examine the expression of all served on purified IFNAR KO B cells treated with the above known HSPGs (syndecan 1–4, glypican 1–6, TGF-bR3, agrin, and stimulatory ligands (Fig. 4, dashed lines). Altogether, our results CD44v3). Their expression in naive and IFN-I–treated B cells was indicate that a variety of stimuli normally present during the examined by reverse transcription quantitative-PCR and surface

FIGURE 4. Both BCR- and TLR-mediated signaling upregulate HS on B cells. Splenic B cells were purified from C57BL/6 or IFNAR KO mice and incubated for 24 h with (A) media alone, (B) anti-IgM Ab (2 mg/ml), (C) LPS (1 mg/ml), (D) type A CpG oligonucleo- tide (5 mg/ml), or (E) type B CpG oligonu- cleotide (5 mg/ml). Shaded histograms show staining with an isotype control. The y-axis for all histograms is percent of maximum. All histograms are representative experiments, using one to two mice, and were repeated at least five times. 5544 UPREGULATION OF HS ON B CELLS UPON VIRAL INFECTION staining (when commercial Abs were available). We observed that later, HS expression on B cells was assessed by flow cytometry. whereas most HSPGs were expressed at extremely low levels or Surprisingly, we observed that HS induction was not affected by not expressed at all (data not shown), syndecan 4 was readily the absence of syndecan 4 expression on B cells (Fig. 5C). detectable. However, its surface expression was only slightly in- To take an unbiased approach in identifying HSPGs expressed on creased in B cells cultured in the presence of IFN-I compared with B cells, we performed a Western blot for HS on lysates isolated that in freshly isolated naive B cells (Fig. 5A). To determine if from ex vivo or IFN-I–treated B cells (using the 3G10 Ab). In syndecan 4 is modified with HS, ex vivo and IFN-I–treated B cells agreement with our above results, HS expression was not detected were treated with or without heparinase (an enzyme that degrades on ex vivo B cells (Fig. 5D). However, in IFN-I–treated wt B cells, HS). Cells were then lysed, and syndecan 4 was immunoprecipi- we observed three bands corresponding with the molecular mass tated. This was followed by a Western blot for HS using the 3G10 of ∼130, 55, and 37 kDa. Two of these bands (55 and 37 kDa) Ab (Seikagaku), which recognizes a neo-epitope that is generated were absent in IFN-I–treated syndecan 4 KO B cells, suggesting upon heparinase treatment. Fig. 5B shows that syndecan 4 from that these two lower molecular mass bands correspond with syn- IFN-I–treated B cells, but not from naive B cells, is associated decan 4. This is in agreement with the molecular mass of HS- with HS (compare heparinase treated ex vivo and IFN-I treated modified syndecan 4 observed in Fig. 5B. The presence of a 130- lanes), indicating that syndecan 4 is modified with HS in response kDa band in IFN-I–treated wt and syndecan 4-deficient B cells to IFN-I treatment. suggests that HS moieties are also carried by an additional high To test if syndecan 4 is the sole HSPG that accounts for HS molecular mass HSPG. Notably, culturing of B cells in the ab- surface expression on B cells, we generated syndecan 4 KO bone sence of IFN-I induced only HS-modified syndecan 4 (Supple- marrow chimeras by i.v. injection of syndecan 4 KO bone marrow mental Fig. 2). Only when B cells were cultured in the presence Downloaded from (CD45.2+) into lethally irradiated congenic wt mice (CD45.1+). of IFN-I, or isolated from a poly I:C-injected mouse, were both Upon reconstitution, we confirmed that syndecan 4 was not syndecan 4 and the 130-kDa HSPG induced. These data indicate expressed in CD45.2+ B cells by RT-PCR (data not shown). that various stimuli can induce HS-modified syndecan 4, but IFN-I B cells were purified from wt and syndecan 4 KO bone marrow signaling induces HS modification of both syndecan 4 and the chimeras and treated in culture with IFN-I. Twenty-four hours 130-kDa HSPG. http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. Syndecan 4 is modified with HS on B cells upon IFN-I treatment. A, Purified B cells from C57BL/6, either ex vivo (solid line) or treated with IFN-I for 24 h (dashed line), were stained for syndecan 4 and analyzed by flow cytometry. Isotype control is represented by shaded histogram. B, Purified B cells, either cultured for 24 h in the presence of IFN-I or isolated immediately ex vivo, were treated with or without heparinase III and chondroitinase before being lysed and immunoprecipitated for syndecan 4. Immunoprecipitates were then run on an SDS-PAGE gel before blotting for HS (3G10). Arrows indicate both syndecan 4 bands. Ab H chain (HC) and L chain (LC) are indicated. C, Splenic B cells were purified from either C57BL/6 (solid line) or syndecan 42/2 bone marrow chimeras (dashed line) and cultured in the presence of IFN-I. Twenty-four hours after IFN-I treatment, cells were harvested and stained for HS (populations gated on CD45.2+). Isotype control is represented by shaded histogram. D, Purified ex vivo or IFN-I–treated B cells were treated with or without heparinase and chondroitinase prior to lysis and Western blot for HS (clone 3G10; Seikaguku). The y-axis for all histograms is percent of maximum. Numbers on blot indicate kDa molecular mass marker. Histograms and Western blot are representative experiments, using one to two mice, and were performed at least three times. The Journal of Immunology 5545

HS expression on B cells is essential for APRIL-mediated a combination of both. To investigate the mechanism of HS up- signaling regulation on B cells, we examined both transcriptional and surface Given the role of HS in receptor signaling, we hypothesized that expression (when commercial Abs were available) of all known HS induction at the B cell surface increases responsiveness to HSPGs. Of all HSPGs assessed, only syndecan 4 was expressed at HS-binding cytokines (2). To address this hypothesis, we analyzed significant levels. Its expression was only slightly enhanced upon the signaling of APRIL, a member of the TNF family that has treatment of B cells with IFN-I (Fig. 5A). However, we found that been shown to bind HS (12, 20). APRIL promotes B cell survival the amount of HS associated with syndecan 4 was dramatically and IgG to IgA isotype switching (13). We treated purified B cells increased in B cells that had been exposed to IFN-I (Fig. 5B). with soluble APRIL (produced by transfection of 293T cells) Notably, we also detected the presence of a high molecular mass or with control media (supernatant from pcDNA3.1-transfected HSPG (around 130 kDa; Fig. 5D). As this HSPG was only induced 293T cells), in the presence or absence of IFN-I and heparinase upon IFN-I signaling (Supplemental Fig. 2), the identity of this either individually or concomitantly. We then assessed APRIL HSPG may give clues as to the function of HS upregulation on signaling by measuring secreted IgA. As shown in Fig. 6A,we B cells in response to IFN-I signaling. The tendency of HSPGs to found that heparinase-treated B cells produced less IgA than oligomerize or to run above their predicted molecular mass (23) untreated controls. IFN-I treatment, which we show induces HS makes it difficult to identify the nature of this HSPG. expression, increased APRIL-mediated IgA production. Simulta- We were surprised to find that HS expression was similar in neous addition of heparinase and IFN-I drastically reduced APRIL- IFN-I–treated wt and syndecan 4 KO B cells (Fig. 5C) given induced IgA production. Importantly, B cell responsiveness to that syndecan 4 is modified with HS upon IFN-I treatment (Fig. APRIL, via HS upregulation, was enhanced upon IFN-I signaling, 5B,5D). One possibility is that the contribution of syndecan 4 Downloaded from as IgA production was reduced in IFNAR KO B cells (Fig. 6B). to HS surface display is minor compared with that of the 130-kDa Altogether, these results indicate that IFN-I–induced HS expres- HSPG. Another possible explanation is that the synthesis of the sion on B cells is important for in vitro APRIL responsiveness. mature form of HS containing N-sulfated glucosamine residues, or its detection by the Ab (epitope detected by the 10E4 Ab used Discussion in our flow cytometry experiments) is limiting in B cells under

In this study, we present evidence that HS expression is tightly conditions that trigger a high level of HS (such as ex vivo IFN-I http://www.jimmunol.org/ regulated in mature B cells in vivo: whereas HS is barely detectable treatment; Fig. 5C). In the absence of syndecan 4, there would in naive splenic B cells, its expression was upregulated by the thus be an increased amount of HS linked to the high molecular action of IFN-I in mice. Additionally, we observed that HS ex- mass HSPG, leading to similar levels of overall surface HS in wt pression was induced upon stimulation of murine splenic B cells and syndecan 4 KO B cells. via TLR-dependent pathways as well as BCR stimulation. Im- IFN-I treatment induces an upregulation of HS modification portantly, we did not observe an increase in HS expression on the on syndecan 4 molecules (Fig. 5B,5D), suggesting that IFN-I acts non-B cell population after viral infections or poly I:C injection on HS biosynthesis. There are multiple steps where this may occur, (Fig. 3B). This does not appear to be due to an inability of these as HS biosynthesis involves numerous enzymes (24). Initially, the cells to express HS, as ex vivo activation of monocytes and CD4 proteoglycan is modified with a tetrasaccharide linker to which HS by guest on September 28, 2021 T lymphocytes increases HS expression at the cell surface (21, will be extended. This process is initiated by the XYLT enzyme 22). Altogether, these results indicate that HS is poorly expressed family. Upon completion of the tetrasaccharide linker, the en- on naive B cells, but its expression can be upregulated on lym- zymes EXTL2 and EXTL3 perform the addition of the first N- phocytes upon exposure to a variety of stimuli normally present acetyl glucosamine, thereby dictating that the chain will be HS during the course of an infection. To our knowledge, our study is and not chondroitin sulfate. This is followed by disaccharide chain the first to show that HS is upregulated on immune cells in vivo. polymerization by the exostosin enzymes EXT1 and EXT2. Fi- Our results are likely to be applicable to other species: van der nally, the disaccharide chain is heterogeneously deacetylated and Voort et al. (15) demonstrated that activation of ex vivo B cells, sulfated by the NDST enzyme family. To determine if HS syn- via BCR or CD40, enhances HS expression on human tonsillar thesis was upregulated, we assessed the expression of HS- B cells. Such dynamic regulation of HS expression suggests that synthesizing enzymes by reverse transcription quantitative-PCR HS may play a critical role in regulating immune function upon in ex vivo and IFN-I–treated B cells. Although we did not see infection. an increase in the mRNA levels of the HS-polymerizing enzymes We hypothesized that the induction of HS could be mediated (EXT1 and EXT2), we did see a 2-fold induction in EXTL2, through upregulation of HSPGs, increased synthesis of HS, or EXTL3, XYLT1, NDST1, and NDST2 (data not shown).

FIGURE 6. IFN-I treatment increases B cell responsiveness to APRIL in an HS-dependent manner. Puriﬁed splenic B cells from wt (A) or IFNAR KO (B) mice were incubated with supernatant from APRIL- or pcDNA3.1-transfected 293T cells. B cells were either left untreated or were treated with heparinase III, IFN-I, or both. IgA production was measured by ELISA after 6 d of culture. Both graphs are experimental averages from two to three experiments where one to two mice of speciﬁed genotype were used per experiment. *p , 0.05, ***p , 0.0005. 5546 UPREGULATION OF HS ON B CELLS UPON VIRAL INFECTION

Intriguingly, we also observed a 10-fold decrease in EXTL1 (data regulation of the Ab responses). Furthermore, if IFN-I–mediated not shown). Whether or not this modest collective increase in HS induction on B cells enhances their responsiveness, this may expression of these enzymes contributes to the observed increase have implications in the understanding of autoimmune disorders in surface HS remains to be determined. It is also possible that with a B cell component. For example, systemic lupus erythe- HS-synthesizing enzymes are regulated posttranslationally, and matosus, an autoimmune disorder characterized by elevated IFN-I this could contribute to the increased HS in IFN-I–treated B cells. and autoreactive B cells (35), may be better understood as a result APRIL, a member of the TNF family of ligands, is implicated of the work presented in this study. in B cell survival, isotype switching, and T cell-independent Ab responses (13, 14, 25). We have shown that IFN-I treatment of Acknowledgments B cells increased IgA production in response to APRIL in an HS- We acknowledge the Center for Host-Pathogen Studies Mouse/Virus core dependent manner. We observed that upon culturing, HS expres- for assistance with viral stocks. sion at the surface of B cells spontaneously increases (Fig. 3C, media). This likely helps explain why APRIL signaling can be Disclosures measured in cultured B cells even in the absence of IFN-I or other The authors have no financial conflicts of interest. stimulators of HS expression (12). In addition to regulating cytokine responsiveness, HS is involved in a broad variety of biological processes including cell adhesion References and migration (2). It would be interesting to determine if HS 1. Bernfield, M., M. Go¨tte, P. W. Park, O. Reizes, M. L. Fitzgerald, J. Lincecum, and M. Zako. 1999. Functions of cell surface heparan sulfate proteoglycans. upregulation in response to IFN-I production has a direct impli- Annu. Rev. Biochem. 68: 729–777. Downloaded from cation on other aspects of B cell biology. For example, during an 2. Bishop, J. R., M. Schuksz, and J. D. Esko. 2007. Heparan sulphate proteoglycans immune response, lymphocyte egress from lymphoid organs is fine-tune mammalian physiology. Nature 446: 1030–1037. 3. Lortat-Jacob, H. 2009. The molecular basis and functional implications of temporarily shut down in response to several mediators including chemokine interactions with heparan sulphate. Curr. Opin. Struct. Biol. 19: 543– IFN-I. The role of IFN-I on lymphocyte retention is explained in 548. 4. Lin, X. 2004. Functions of heparan sulfate proteoglycans in cell signaling during part by inhibition of sphingosine 1-phosphate receptor-1 by CD69 development. Development 131: 6009–6021.

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IFN-I

100 media CpG

0 100 101 102 103 104

TACI

! Supplemental Figure Legends

Supplemental Figure 1. HS expression on B cells is not due to TACI binding HS- bearing proteins.

Purified splenic B cells were cultured for 24 hours in media alone (dashed line), IFN-I

(thin solid line), or CpG (thick solid line). After 24 hours, B cells were stained for TACI with PE-conjugated anti-TACI. Isotype control is represented as shaded histogram. Y axis is percent of maximum. Histogram is a representative experiment, using 1-2 mice, repeated at least 3 times.

Supplemental Figure 2. IFN treatment of B cells or injection of poly I:C induce both

SDC4 and 130 kDa HSPG, while culturing of B cells in the absence of IFN only induces SDC4.

Purified splenic B cells were cultured ± IFN-I for 24 hours before being treated with heparanase and chondroitinase prior to lysis (lanes 1 and 2). B cells were purified from mice injected (iv) with either PBS (lanes 3 and 4) or poly I:C (lanes 5 and 6) 24 hours prior. B cells were then treated ± heparanase and chondroitinase prior to lysis. Lysates were then run on a 10% SDS-PAGE gel and blotted for heparan sulfate using the 3G10 antibody. Western blot is a representative experiment repeated twice.